D  M  KLU  T^M  C.  U I K* TAj^-tTl  D  K  ft  T 

742  NOn^SROADWAY 
kNGELl 

Issued  April  8, 1913. 

U.  S.  DEPARTMENT  OF(AGRICULTURE, 

BUREAU  OF(JANIMAL  INDUSTRY.— Bulletin  162. 

A.  D.  MELVIN,  Chief  of  Bureau. 


m  :ACT0RS  INFLUENCING  THE  CHANGE  IN 
FLAVOR  IN  STORAGE  BUTTER. 


BY 


LA.  CO.^E^ALASSIl 
634  SOiPtfW&TLAKE  AVE. 


L.  A.  ROGERS,  Bacteriologist;  W.  N.  BERG,  Chemist; 
C.  R.  POTTEIGER,  Assistant  Chemist, 

AND 
B.  J.  DAVIS,  Assistant. 


X 


'% 


% 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1913. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melvin. 

Assistant  Chief:  A.  M.  Farrington. 

Chief  Cleric:  Charles  C.  Carroll. 

Animal  Husbandry  Division:  George  M.  Rommel,  chief. 

Biochemic  Division:  M.  Dorset,  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Field  Inspection  Division:  R.  A.  Ramsay,  chief. 

Meat  Inspection  Division:  R.  P.  Steddom,  chief. 

Pathological  Division:  John  R.  Mohler,  chief. 

Quarantine  Division:  Richard  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schroeder,  superintendent. 

Editor:  James  M.  Pickens. 

DAIRY  DIVISION. 

B.  H.  Rawl,  chief. 

Helmer  Rabild,  in  charge  of  Dairy  Farming  Investigations. 

S.  C.  Thompson,  in  charge  of  Dairy  Manufacturing  Investigations. 

L.  A.  Rogers,  in  charge  of  Research  Laboratories. 

Ernest  Kelly,  in  charge  of  Market  Milk  Investigations. 

Robert  McAdam,  in  charge  of  Renovated  Butter  Inspection. 


Issued  April  8,  1913. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  162. 

A.  D.  MELVIN,  Chief  of  Bureau. 


FACTORS  INFLUENCING  THE  CHANGE  IN 
FLAVOR  IN  STORAGE  BUTTER. 


BY 


L.  A.  ROGERS,  Bacteriologist;  W.  N.  BERG,  Chemist; 
C.  R.  POTTEIGER,  Assistant  Chemist, 

AND 


B.  J.  DAVIS,  Assistant. 


LIBRARY  OF  THE 
LA.  CO.  MEDICAL ASSft 

634  SOUTH  WESTLAKE  AVE. 
LOS  ANPF1FS 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1913. 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 
Washington,  D.  C,  October  8,  1912. 
Sir:  I  have  the  honor  to  transmit  herewith  for  publication  in  the 
bulletin  series  of  this  bureau  a  manuscript  entitled  "Factors  Influ- 
encing the  Change  in  Flavor  in  Storage  Butter,"  by  Messrs.  L.  A. 
Rogers,  W.  N.  Berg,  C.  R.  Potteiger,  and  B.  J.  Davis,  of  the  Dairy 
Division. 

Respectfully, 

A.  D.  Melvin, 
Chief  of  Bureau. 
Hon.  James  Wilson, 

Secretary  of  Agriculture. 


CONTENTS, 


Page. 

Introduction 5 

Possible  causes  of  change 6 

Proteolysis  in  butter 9 

Previous  work 9 

Analytical  difficulties 9 

Results  by  Gray  and  others 11 

Objections  to  ferric  chlorid  and  tannic  acid  as  protein  precipitants 14 

New  method  for  detecting  proteolysis  in  butter 16 

The  influence  of  sodium  chlorid  on  the  precipitability  of  casein  by 

acetic  acid 16 

Method  for  the  estimation  of  water-soluble  nitrogen  in  butter 18 

Description  of  samples 23 

Discussion  of  results,  Table  2 25 

Conclusions 25 

Proteolysis  in  milk 26 

Possible  objection  to  the  new  method  for  detecting  proteolysis  in  butter. . .  26 
The  inhibiting  effect  of  sodium  chlorid  and  cold  storage  upon  the  activity 

of  galactase  in  buttermilk •  27 

Method  of  measuring  the  activity  of  galactase  in  buttermilk 28 

Results 28 

The  inhibiting  effect  of  sodium  chlorid  and  cold  storage  upon  the  activities 

of  proteolytic  enzyms  in  sterilized  skim  milk 30 

Description  of  samples 30 

Results 31 

Conclusions 31 

The  indirect  action  of  bacteria 32 

Reinoculation  of  cream * 33 

The  possible  oxidation  of  butter  by  inclosed  air 34 

Method  of  gas  analysis 35 

Results t 36 

The  effect  of  metals  on  butter 38 

Earlier  investigations 38 

Method  of  analysis 42 

Relation  of  iron  in  butter  to  iron  in  the  cream 43 

Distribution  of  iron  between  fat  and  curd  solution 44 

The  influence  of  iron  on  flavor 45 

The  influence  of  copper  on  flavor 48 

Contamination  of  cream  with  iron  from  containers 50 

Theoretical  considerations 55 

The  oxidation  of  lactose  in  butter 57 

Description  of  samples 58 

Methods  and  experimental  procedure 58 

The  possible  oxidation  of  lactose  in  storage  butter.by  a  peroxid 61 

Odors  produced  in  milk  by  the  addition  of  iron  salts 64 

The  production  of  iodoform-reacting  substances  in  milk  by  ferrous  iron 66 

Summary 68 

3 


ILLUSTRATION 


Page. 
Fig.  1. — Apparatus  used  for  obtaining  the  gas  from  a  can  of  flutter 35 

4 


FACTORS  INFLUENCING  THE  CHANGE  IN  FLAVOR  IN 
STORAGE  BUTTER. 


INTRODUCTION. 

The  economic  conditions  in  this  country  which  have  made  it  neces- 
sary to  hold  butter  in  storage  for  long  periods  have  increased  the 
importance  of  the  changes  that  take  place  in  butter  on  standing. 
A  change  that  passes  unnoticed  in  butter  that  is  used  when  a  week  or 
two  old  may  become  a  serious  defect  after  three  or  four  months  in 
storage.  The  great  variation  and  complexity  of  the  changes  in  flavor 
indicate  a  corresponding  complexity  in  the  chemical  alteration  in  the 
butter,  and  while  it  is  true  that  some  of  the  modifications  are  well 
known  it  is  becoming  evident  that  the  various  flavors  are  produced 
by  changes  too  small  to  be  measured  by  the  ordinary  methods  of  the 
laboratory.  Under  certain  circumstances  free  fatty  acids  may  be 
formed,  a  condition  usually  associated  with  a  rancid  flavor.  How- 
ever, it  is  evident  that  the  fatty  acids  alone  are  not  the  cause  of  the 
rancid  flavor,  since,  in  the  process  of  renovating,  the  rancid  flavor 
is  removed  while  a  large  part  of  the  acid  remains. 

It  is  possible  that  the  flavor-giving  substances  are  produced  in  very 
small  quantities  and  that  their  formation  is  not  necessarily  connected 
with  or  in  proportion  to  the  grosser  changes  measurable  by  the  ordi- 
nary analytical  methods.  There  are  several  substances  in  butter 
that  are  more  or  less  unstable  under  ordinary  circumstances,  i.  e.,  the 
proteins  of  milk  in  their  hydrated  condition,  lecithin,  citric  acid,  lac- 
tic acid,  and  other  products  of  bacterial  action.  But  little  work  has 
been  done  in  which  the  storage  flavor  was  shown  to  be  related  to 
chemical  changes  involving  any  of  these  substances.  This  is  proba- 
bly due  to  the  fact  that  while  butter  fat  is  easily  handled  for  analyti- 
cal purposes,  it  is  difficult  to  separate  from  the  butter  fat  the  other 
fatlike  substances,  such  as  lecithin.  The  remaining  part  of  the 
butter,  which  will  be  called  the  butter  curd  solution,  is  of  such  a  physi- 
cal consistency  that  it  can  not  very  well  be  used  directly  for  quanti- 
tative analytical  work. 

In  considering  the  problem  of  storage  flavor,  its  causes,  and  the 
methods  of  studying  the  problem,  it  is  well  to  bear  in  mind  one  or 
two  of  the  facts  involved  in  the  physiology  of  the  senses  of  taste  and 
smell.  It  is  well  known  that  several  different  substances  may  taste 
alike:  Thus  sugar,  saccharin,  lead  acetate,  glycerin,  and  perhaps 
still  other  substances,  all  taste  sweet.     Chemically  they  are  not  at 

5 


6  CHANGE   IN   FLAVOR  OF  STORAGE  BUTTER. 

all  similar.  While  trimethylamin  may  be  the  specific  cause  of  fishy 
flavor  in  herring  brine,  it  is  not  necessarily  the  cause  of  fishy  flavor 
in  butter.  It  is  possible,  reasoning  by  analogy,  that  many  different 
substances  may  cause  "fishy"  flavor. 

The  sense  of  smell  is  very  delicate  and  can  detect  astonishingly 
small  amounts  of  material,  so  small  that  the  most  delicate  balances 
could  not  weigh  them.  A  flavor  is  a  mixed  sensation  in  which  the 
sense  of  taste  and  smell  take  a  leading  part.  Howell x  states  that 
0.00005  grams  of  quinine  in  100  cubic  centimeters  of  water  is  detecti- 
ble  on  the  root  of  the  tongue.  "It  is  recognized  in  chemical  work, 
for  instance,  that  traces  of  known  substances  too  small  to  give  the 
ordinary  chemical  reactions  may  be  detected  easily  by  the  sense  of 
smell.  According  to  the  experiments  of  Fischer  and  Penzoldt,  mer- 
captan  may  be  detected  in  a  dilution  of  460,000,000  °f  a  milligram 
in  50  cubic  centimeters  of  air."  * 

While  the  off  flavors  of  butter  may  not  be  caused  by  the  formation 
of  such  inconceivably  small  amounts  of  odoriferous  substances,  yet 
such  data  are  of  practical  significance  in  so  far  as  they  indicate  that 
the  analytical  method  of  studying  storage  flavors  may  be  wholly 
inadequate.  There  may  be  many  substances  2  whose  isolation  or 
detection  in  butter  might  be  very  difficult  if  not  impossible  by  present 
methods,  and  which  would  still  impart  to  the  butter  sufficient  odor 
and  taste  to  be  distinctly  perceptible. 

POSSIBLE  CAUSES  OF  CHANGE. 

The  marked  influence  of  bacteria  on  the  flavor  of  milk,  cheese,  and 
other  dairy  products  naturally  leads  to  the  conclusion  that  the  same 
organisms  would  be  an  important  if  not  the  only  factor  concerned  in 
the  changes  in  butter.  It  has  been  demonstrated,  particularly  by  the 
work  of  Jensen,3  that  under  certain  conditions  bacteria  multiply  in 
butter  and  have  a  direct  influence  on  the  flavor  of  the  product. 

It  should  be  remembered,  however,  that  the  butter  on  which 
Jensen  and  other  European  investigators  worked  differs  in  one  very 
essential  particular  from  the  ordinary  American  butter.  While  the 
salt  content  of  most  European  butter  is  low  enough  to  permit  the 
growth  of  bacteria,  American  butter  contains  sufficient  salt  to  bring 
its  concentration  in  the  water  of  the  butter  to  18  per  cent  or  more. 
It  is  to  be  expected  that  bacteria  would  not  grow — or  at  least  would 

1  Howell,  William  H.    Textbook  of  Physiology.    Philadelphia,  1906.    See  pp.  275-280. 

2  Zwaardemaker,  H.  Geruch.  Ergebnisse  der  Physiologie.  Abteilung  2,  vol.  1,  pp.  896-909.  Wies- 
baden, 1902. 

3  Jensen,  Orla.  Bakteriologische  Studien  uber  danische  Butter.  Centralblatt  fur  Bakteriologie,  Para- 
sitenkunde  und  Infektionskrankheiten.    Abteilung  2,  vol.  29,  no.  23/26,  pp.  610-616.    Jena,  Apr.  8,  1911._ 

Jensen,  Orla.  Studien  uber  das  Ranzigwerden  der  Butter.  Centralblatt  fur  Bakteriologie.  Parasi- 
tenkunde  und  Infektionskrankheiten.  Abteilung  2,  vol.  8,  no.  1,  pp.  11-16,  Jan.  4;  no.  5,  pp.  140-144,  Feb. 
5;  no.  6,  pp.  171-174,  Feb.  10;  no.  7,  pp.  211-216,  Feb.  17;  no.  8,  pp.  248-252,  Feb.  24;  no.  9,  pp.  278-281, 
Mar.  4;  no.  10,  pp.  309-312,  Mar.  8;  no.  11,  pp.  342-346,  Mar.  13;  no.  12,  pp.  367-369,  Mar.  15;  no.  13,  pp. 
406-409,  Mar.  24.    Jena,  1902. 


POSSIBLE   CAUSES   OF   CHANGE.  7 

grow  only  very  sparsely — under  these  conditions,  and  the  investiga- 
tions in  this  country  confirm  this  supposition.  Rahn,  Brown,  and 
Smith1  found  in  some  samples  of  butter  a  torula  able  to  grow  very  slowly 
in  salt  solutions  at  low  temperatures,  but  this  occurred  in  such  small 
numbers  that  it  could  not  account  for  much  of  the  deterioration  of 
the  butter.  In  our  own  work  we  have  found  no  evidence  of  bacterial 
growth  under  normal  conditions,  with  the  exception  of  a  small  multi- 
plication of  torula  at  high  storage  temperatures.  In  these  cases 
there  was  no  apparent  relation  between  the  growth  of  torula  and 
change  in  flavor.  Moreover,  the  same  changes  took  place  in  dupli- 
cate lots  of  butter  held  at  temperatures  so  far  below  the  freezing 
point  that  there  could  be  no  possibility  of  growth.  Any  flavors  that 
appear  in  butter  may  be  found  in  butter  held  at  the  commercial 
storage  temperature  of  zero  or  below  (Fahrenheit),  and  any  explana- 
tion of  the  cause  of  these  changes  which  does  not  take  this  fact  into 
consideration  is  obviously  fallacious,  or  at  best  valid  for  certain  con- 
ditions only. 

In  some  of  our  earlier  work2  the  possible  influence  of  lipolytic 
enzyms  was  suggested,  but  it  was  soon  found  that  in  many  cases 
butter  showed  a  marked  change  in  flavor  without  any  appreciable 
hydrolysis  of  the  fat.  This  observation  is  confirmed  by  the  work  of 
Rahn,  Brown,  and  Smith  cited  above.  The  action  of  other  enzyms, 
as,  for  instance,  the  proteolytic  enzym  of  the  milk  or  those  secreted 
by  bacteria,  is  not  necessarily  excluded. 

The  influence  of  the  acidity  of  the  cream  on  the  flavor  of  butter  has 
already  been  pointed  out.3  It  has  also  been  suggested  *  that  a  slow 
oxidation  may  take  place  in  the  interior  of  a  package  of  butter,  due 
to  the  numerous  small  bubbles  of  air  inclosed  in  the  butter.  Even  a 
superficial  examination  of  the  work  already  done  shows  that  the  ques- 
tion is  a  very  complicated  one  and  that  the  difficulties  in  the  way  of  a 
solution  are  many.  In  studying  the  ripening  of  cheese  pronounced 
chemical  changes  are  available  f^r  measuring  the  progress  of  the  ripen- 
ing. In  butter  the  changes  are  scarcely  appreciable.  The  investi- 
gator is  thus  forced  to  rely  on  the  sense  of  taste  and  smell  for  a 
measure  of  the  change.  Some  butter  judges  have  developed  marked 
ability  in  detecting  and  estimating  the  intensity  of  various  flavors, 
but  at  best  the  sense  of  taste  is  uncertain,  and  any  numerical  scale 
based  on  this  faculty  is  necessarily  an  arbitrary  one  and  subject  to 
fluctuation  in  its  value.     Two  butter  judges  can  not  be  expected 

i  Rahn,  Otto,  Brown,  C  W.,  and  Smith,  L.  M.  Keeping  qualities  of  butter.  Michigan  Agricultural 
College  Experiment  Station,  Technical  Bulletin  2.    East  Lansing,  September,  1909. 

2  Rogers,  Lore  A.  Studies  upon  the  keeping  quality  of  butter.  United  States  Department  of  Agricul- 
ture, Bureau  of  Animal  Industry,  Bulletin  57,  Washington,  1904. 

*  Rogers,  L.  A.,  and  Gray,  C  E .  The  influence  of  acidity  of  cream  on  the  flavor  of  butter.  United  States 
Department  of  Agriculture,  Bureau  of  Animal  Industry,  Bulletin  114,  Washington,  1909. 

*  Rogers,  L.  A.  Fishy  flavor  in  butter.  United  States  Department  of  Agriculture,  Bureau  of  Animal 
Industry,  Circular  146,  Washington,  1909. 


8  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

always  to  agree,  because  the  definitions  of  flavors  can  not  be  reduced 
to  exact  terms  and  the  amount  of  deduction  on  the  numerical  scale 
for  various  flavors  can  not  be  fixed. 

The  most  serious  difficulty  in  experimental  work  on  butter  is  in 
cod  trolling  the  conditions  under  which  the  butter  is  made.  So  many 
apparently  unimportant  factors  have  an  influence  on  the  flavor  that 
it  is  nearly  impossible  to  make  butter  with  a  normal  flavor  and  have 
only  one  varying  factor.  The  work  is  further  complicated  by  the 
sequence  of  flavors  that  frequently  occurs  in  butter  held  in  storage. 
It  is  evident  that  the  usual  off  flavors  are  in  many  cases  a  combination 
of  flavors  and  that  the  flavors  themselves  are  caused  by  a  combination 
of  circumstances  and  not  by  a  single  cause.  It  is  probable  also  that 
identical  flavors  may  be  caused  by  different  factors. 

In  the  work  reported  in  this  paper  we  have  attempted  to  determine 
the  part  played  by  certain  factors  in  the  general  change  in  flavor  in 
storage  butter  without  directing  the  investigation  toward  any  par- 
ticular flavor  or  attempting  to  cover  all  of  the  causes  of  deterioration. 

In  this  we  have  been  guided  by  the  previous  work,  which  has  indi- 
cated certain  points  at  which  the  problem  could  be  attacked  with 
some  promise  of  positive  results.  It  has  been  observed,  for  instance, 
that  when  a  lot  of  sweet  cream  is  divided,  one  half  churned  at  once 
and  the  other  half  pasteurized  and  churned,  the  butter  from  the 
unpasteurized  half  deteriorates  very  quickly,  while  the  pasteurized- 
cream  butter  has  exceptionally  good  keeping  qualities.  What  has 
been  removed  by  the  pasteurization  that  has  such  a  marked  influence 
on  the  butter?  The  enzyms  of  the  milk  are  partly  or  entirely 
destroyed  and  a  large  proportion  of  the  bacteria  are  killed.  Are  the 
proteolytic  enzyms  of  the  milk  able  to  work  under  the  conditions 
existing  in  butter  and  have  they  any  influence  on  the  flavor  of  the 
butter?  Is  there  any  appreciable  proteolysis  in  butter  even  under 
favorable  conditions  ?  If  the  two  lots  of  cream  are  ripened,  the  keep- 
ing quality  of  the  butter  from  the  unpasteurized  cream  is  increased, 
while  that  from  the  pasteurized  cream  is  decreased.  In  the  process 
of  ripening  the  bacterial  growth  is  confined  almost  entirely  to  one 
variety,  Taut  it  does  not  necessarily  follow  that  these  bacteria  have 
any  direct  deleterious  action.  The  growth  of  the  bacteria  produces  a 
considerable  quantity  of  acid,  and  the  chemical  instability  of  the 
product  is  increased  accordingly. 

Does  the  air  which,  as  has  been  shown,  is  inclosed  in  the  butter 
effect  an  appreciable  oxidation  ?  Milk  and  cream  is  handled  in  con- 
tainers in  which  it  may  be  exposed  to  tin,  iron,  or  copper.  Under 
these  conditions  it  is  reasonable  to  suppose  that  small  amounts  of  the 
metals,  especially  the  iron  and  copper,  will  be  dissolved  and  carried 
into  the  butter.  Do  the  salts  formed  by  the  metals  with  organic  acids 
of  the  cream  affect  the  flavor  of  the  butter  ? 


PEOTEOLYSIS   IN   BUTTER. 


PROTEOLYSIS  IN  BUTTER. 


It  has  long  been  known  that  butter  differs  from  milk  in  its  compo- 
sition only  in  the  relative  amounts  of  the  constituents  present  in  the 
two.  Among  these  constituents  which  early  attracted  attention  as 
possible  causes  of  storage  flavor  because  of  their  chemical  instability 
were  the  proteins,  mainly  casein.  Proteins  in  the  hydrated  or  moist 
condition  in  the  presence  of  water  are  known  to  be  unstable,  and  it  is 
but  natural  that  these  substances,  wherever  they  may  occur  in  food, 
should  be  looked  upon  as  possible  sources  of  off  flavor.  It  is  almost 
certain  that  in  butter  containing  no  salt,  or  salt  in  an  insufficient 
amount,  or  in  butter  that  is  not  stored  at  sufficiently  low  tempera- 
ture, the  proteins  present  do  undergo  hydrolysis  and  perhaps  putre- 
faction and  other  obscure  changes  as  well.  But  the  present  work 
does  not  concern  itself  with  such  material.  The  problem  is :  If  stor- 
age flavor  develops  in  butter  properly  made  and  properly  stored,  do 
the  proteins  contribute  in  any  way  toward  this  off  flavor  ? 

Certain  conditions  in  butter  favor  proteolytic  changes,  namely,  the 
presence  of  water,  bacteria,  and  of  the  proteolytic  enzym  known  as 
galactase,  which  occurs  normally  in  milk.  Other  conditions,  as  low 
temperature,  the  presence  of  sodium  chlorid,  the  partial  inactivation 
of  the  galactase  by  pasteurization,  tend  to  prevent  or  retard  proteo- 
lytic changes. 

It  has  already  been  shown  by  other  investigators  that  under  con- 
ditions of  comparatively  high  temperature  and  low  salt  the  butter 
proteins  will  undergo  changes.  In  the  present  work  an  attempt  was 
made  to  determine  whether  the  galactase  present  in  butter  made  from 
pasteurized  or  from  unpasteurized  cream  can  digest  casein  in  spite  of 
the  retarding  influence  of  low  temperature  and  high  salt  concen- 
tration. 

PREVIOUS    WORK. 

Analytical  difficulties. — When  butter  is  melted  and  allowed  to  stand, 
the  water  present,  containing  the  salt  and  casein  in  solution  and  in 
suspension,  will  settle  to  the  bottom  of  the  container,  leaving  the 
supernatant  fat  clear.  The  fat  may  be  poured  off  and  filtered  if 
desired  and  at  once  used  for  quantitative  work.  However,  all  of  the 
fat  can  not  be  poured  off,  because  part  of  it  is  thoroughly  mixed  with 
the  particles  of  curd,  so  that  after  the  most  careful  removal  of  fat 
by  decantation  a  considerable  amount  is  still  left.  Some  of  this 
residual  fat  can  be  removed  by  the  addition  of  ether.  This  will  dis- 
solve the  fat  on  the  upper  surface  of  the  curd  solution  and  permit 
more  fat  to  rise;  but  even  three  or  four  such  washings  with  ether  still 
leaves  in  the  curd  solution  a  considerable  quantity  of  fat,  probably 
20  grams  of  fat  in  100  cubic  centimeters  of  curd  solution.  It  is 
obvious  that  such  a  mixture  of  fat,  sodium  chlorid  solution,  and 

66711°— Bull.  162— J  3 2 


10  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

casein  suspension  is  not  very  well  adapted  to  quantitative  work. 
The  material  will  not  filter,  nor  can  small  samples  of  uniform  com- 
position be  easily  withdrawn  from  it. 

In  order  to  study  the  possible  changes  in  the  proteins  of  butter, 
this  is  the  material  to  be  used.  In  principle,  the  method  of  testing 
for  the  presence  of  active  proteolytic  enzyms  in  this  material  is  no 
different  from  that  used  for  other  purposes,  as,  for  instance,  in  tracing 
the  proteolytic  changes  in  ripening  cheese  or  in  animal  tissue  under- 
going autolysis.  At  first  it  would  seem  as  if  there  should  be  no 
difficulty  in  making  the  usual  nitrogen  partition  in  this  curd  solution 
just  as  it  is  made  on  other  viscous  mixtures  that  are  equally  difficult 
to  filter  and  sample. 

Evidently  it  was  at  the  first  step  in  the  nitrogen  partition  that  the 
difficulties  began,  for,  to  the  best  of  our  knowledge,  none  of  the  pre- 
vious investigators  succeeded  in  precipitating  the  casein  in  the  curd 
solution,  filtering  and  determining  nitrogen  in  the  filtrate  or  the  pre- 
cipitate, in  such  a  manner  as  to  enable  the  investigator  to  draw 
safe  conclusions  from  the  analytic  data  thus  obtained.  To  this  state- 
ment there  are  apparent  exceptions.  On  adding  acetic  acid  in  usual 
amounts  to  some  of  the  curd  solution  as  if  it  were  so  much  milk  for 
the  purpose  of  flocculating  the  casein  and  filtering  no  flocculation  is 
seen  to  occur  and  the  mixture  will  filter  so  slowly  as  to  make  quanti- 
tative work  unreliable  for  obvious  reasons.  If  the  curd  solution  be 
diluted  with  water  until  the  casein  can  be  flocculated  by  acetic  acid 
in  usual  amounts,  filtration  is  then  rapid  and  the  filtrate  can  then  be 
used  for  nitrogen  determinations.  The  nitrogenous  substances  in 
butter,  however,  are  about  75  per  cent  casein,  so  that  on  removal  of 
the  casein  there  is  so  little  nitrogen  left  in  the  filtrate  from  the  diluted 
curd  solution  that  the  unavoidable  errors  in  such  work  are  very  large 
when  compared  with  the  analytic  data  obtained.  Still  less  certain 
are  the  results  obtained  on  the  nitrogen  partition  in  such  a  filtrate, 
because  the  total  nitrogen  is  too  small  for  even  that  determination. 

In  order  to  avoid  the  introduction  of  comparatively  large  errors, 
we  made  many  attempts  to  increase  the  amount  of  curd  solution  used 
and  to  reduce  the  dilution  before  adding  the  precipitant.  Acetic  or 
other  acids  evidently  were  not  previously  used  in  quantities  sufficient 
to  flocculate  the  casein.  Other  precipitants,  such  as  ferric  chlorid, 
phosphotungstic  acid,  tannic  acid,  copper  sulfate,  etc.,  were  tried. 
When  added  to  curd  solutions  diluted  with  but  two  volumes  of  water, 
these  precipitants  will  thoroughly  flocculate  the  protein  and  give  a 
filtrate  that  is  clear,  comes  through  rapidly,  and  can  be  used  for 
quantitative  work.  Unfortunately,  these  precipitants  remove  from 
the  curd  solution  practically  all  of  the  nitrogen,  leaving  too  little  in 
the  filtrate.  The  water-soluble  nitrogen  in  good  butter  is  approxi- 
mately one-fifth  to  one-tenth  of  the  total,  and  in  so  far  as  the  total 


PKOTEOLYSIS   IN   BUTTER  11 

nitrogen  is  represented  by  1  per  cent  of  curd,  or  about  0.1  to  0.2  per 
cent  of  nitrogen,  it  is  necessary  to  use  large  amounts  of  curd  solution 
for  these  precipitations  in  order  that  the  filtrates  may  contain  suffi- 
cient nitrogen  for  accurate  determinations. 

Results  by  Gray  and  others. — Several  years  ago  (1906)  Mr.  C.  E. 
Gray,  then  connected  with  the  Dairy  Division,  began  a  study  of  the 
possible  proteolytic  changes  in  storage  butter  and  their  relation  to  the 
change  in  flavor.  Following  is  his  method  of  making  the  partition 
of  nitrogen  in  butter: 

Total  nitrogen. — Introduce  10  grams  of  butter  into  a  Kjeldahl  flask,  digest,  and 
distill  as  usual. 

To  obtain  nitrogen  in  other  forms:  Melt  2  kilos  of  butter  in  a  hot-water  jacketed 
funnel,  temperature  about  80°  C.  The  melted  butter  is  allowed  to  run  into  a  cream 
separator  with  a  special  bowl  having  a  capacity  of  700  cubic  centimeters  without  milk 
outlets.  This  was  just  large  enough  to  hold  all  of  the  curd  solution  plus  a  small  amount 
of  fat.  As  the  butter  was  fed  in  the  bowl  soon  became  filled  and  the  excess  of  butter 
fat  ran  out,  leaving  the  curd  solution  in  the  bowl.  The  larger  part  of  the  fat  was 
washed  out  by  feeding  gasoline  into  the  bowl.  The  last  portion  of  gasoline  was 
removed  by  feeding  in  water.  The  addition  of  water  was  stopped  as  soon  as  the  out- 
flowing liquid  carried  particles  of  casein.  In  this  way  the  curd  solution  in  2,000  grams 
of  butter  was  separated  from  most  of  the  fat.  The  contents  of  the  bowl  were  trans- 
ferred to  a  1-liter  flask,  25  cubic  centimeters  of  10  per  cent  ferric  chlorid  solution 
were  added,  and  the  total  volume  made  up  to  the  mark.  The  mixture  was  filtered  on 
a  32-centimeter  filter  and  the  faintly  colored  filtrate  used  in  the  following  determina- 
tions. Although  but  600  to  700  cubic  centimeters  of  filtrate  were  obtained,  the 
results  on  aliquot  portions  were  always  calculated  to  1,000  cubic  centimeters. 

It  is  obvious  that  these  filtrates  contained  only  nitrogen  not  pre- 
cipitated by  ferric  chlorid;  that  is,  nitrogen  largely  in  the  form  of 
amino  acids  and  ammonia,  Certain  peptones  are  precipitated  by 
ferric  chlorid.1 

Total  soluble  nitrogen. — Transfer  50  cubic  centimeter  portions  of  the  filtrate  (cor- 
responding to  100  grams  of  butter)  to  Kjeldahl  flasks  and  determine  total  nitrogen. 

"Amino  and  ammonia  nitrogen." — Transfer  a  200  cubic  centimeter  portion  of  the 
ferric  chlorid  filtrate  to  a  300  cubic  centimeter  volumetric  flask.  Add  1  gram  of 
sodium  chlorid  and  sufficient  12  per  cent  tannic  acid  solution  for  maximal  precipi- 
tation. Three  or  four  cubic  centimeters  were  usually  required.  Make  up  to  the  mark 
with  distilled  water,  filter,  and  determine  total  nitrogen  in  100  cubic  centimeter 
portions  of  the  filtrate,  each  of  which  corresponds  to  133$  grams  of  butter. 

"Ammonia  nitrogen."— The  method  described  by  Van  Slyke  and  Hart  2  was  used. 

Transfer  100  cubic  centimeters  of  the  ferric-chloiid  filtrate  (corresponding  to  200 
grams  of  butter)  to  a  Kjeldahl  flask,  add  2  grams  of  magnesium  oxid,  and  boil  for  about 
1$  hours,  catching  the  distillate  in  N/20  acid.  The  excess  of  acid  was  titrated  in  the 
usual  way. 

1  Siegfried,  M.  Zur  Kenntniss  der  Phosphorfleischsaure.  Zeitschrift  fur  Physiologische  Chemie,  vol.  21, 
no.  5/6,  pp.  360-379.    Strassburg,  Apr.  2, 1896. 

Siegfried,  M.  Ueber  Antipepton.  Zeitschrift  fur  Physiologische  Chemie,  vol.  27,  no.  4/5,  pp.  335-347. 
Strassburg,  June  24, 1889.    See  p.  342. 

2  Van  Slyke,  L.  L.,  and  Hart,  E.  B.  Methods  for  the  estimation  of  the  proteolytic  compoimds  con- 
tained In  cheese  and  milk.  New  York  Agricultural  Experiment  Station,  Bulletin  215,  Geneva,  September, 
1902. 


12  CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 

Amino  nitrogen  is  the  difference  between  the  sum  of  the  amino  and  ammonia 
nitrogen  and  the  ammonia  nitrogen. 

Proteose  and  peptone  nitrogen  is  the  difference  between  the  total  soluble  nitrogen 
and  the  sum  of  the  amino  and  ammonia  nitrogen. 

This  method  of  studying  the  distribution  of  nitrogen  in  butter  was 
used  by  Gray  from  1906  to  1907  on  a  very  large  number  of  samples 
of  butter  churned  from  ripened,  unripened,  pasteurized,  and  unpas- 
teurized cream  and  stored  at  various  temperatures.  The  plan  of  the 
investigation  was  very  comprehensive.  It  aimed  to  ascertain  the 
best  conditions  for  the  production  of  butter  of  best  keeping  quality 
and  the  chemical  changes  causing  the  off  flavors  of  storage  butter. 
This  method  was  also  used  by  us  on  one  series  of  24  samples  in  the 
spring  of  1908. 

Gray's  method  of  removing  most  of  the  fat  from  the  butter  by  the 
use  of  the  centrifuge  was  an  improvement,  without  doubt.  But  for 
reasons  to  be  made  apparent  presently  the  analytic  data  obtained 
by  this  method  were  not  regarded  as  conclusive.  More  accurate 
data,  it  is  believed,  were  later  obtained  by  a  method  that  is  free  from 
some  of  the  objections  that  might  be  made  to  the  method  as  originally 
devised  by  Gray. 

In  Table  1  are  some  results  obtained  by  Gray.  The  butter  was 
obtained  from  one  lot  of  cream  which  was  divided  into  eight  portions 
from  which  eight  separate  churnings  were  made.  The  eight  lots  of 
butter  were  packed  in  20-pound  tubs  and  stored  soon  after  churning, 
at  —10°  F.  (—23°  C).  Analyses  were  made  on  the  fresh  butter, 
representing  the  condition  of  the  nitrogen  in  the  butter  before  storage. 
The  two  following  series  of  analyses  were  made  on  the  butter  after 
different  periods  in  storage.  Two  similar  series  of  results -were 
obtained  by  Gray  on  portions  of  the  same  lots  of  butter,  stored  at 
10°  F.  ( - 12°  C.)  and  at  32°  F.  (0°  C).  The  figures  are  not  given 
here,  but  are  in  general  similar  to  those  in  Table  1. 

From  the  results  obtained  by  Gray  it  would  seem  that  in  the 
samples  of  butter  examined  slow  proteolytic  changes  took  place  during 
storage.  At  least  this  is  the  inference  to  be  drawn  on  the  assump- 
tion that  the  method  of  obtaining  the  chemical  data  was  free  from 
avoidable  errors. 


PEOTEOLYSIS   IN   BUTTER. 


13 


Tabi>e  1. — Changes  in  the  distribution  of  nitrogen  in  butter  during  cold  storage 
(-10°  F.,  -23°  0.). 


Butter 
No. 

Age  of 
sample. 

Total 
nitrogen . 

Total 

soluble 

nitrogen. 

Proteose 

and 
peptone 
nitrogen. 

Amino 
nitrogen. 

Aii.monia 
nitrogen. 

10.311 
10.312 
10.313 
10.314 
10.321 
10.322 
10.323 
10.324 

Days. 

0 
206 
298 

0 
206 
298 

0 
206 
298 

0 
206 
298 

0 
206 
298 

0 
206 
298 

0 
206 
298 

0 
206 
298 

Per  cent. 
0.136 

Per  cent. 
0.0039 
.0070 
.0083 
.0041 
.0065 
.0074 
.0053 
.0067 
.0071 
.0045 
.0061 
.0090 
.0020 
.0037 
.0073 
.0030 
.0053 
.0035 
.0037 
.0047 
.0061 
.0065 
.0081 
.0087 

Per  cent. 
0.0013 
.0032 
.0043 
.0026 
.0035 
.0026 
.0003 
.0032 
.0041 
.0005 
.0023 
.0046 

Per  cent. 
0.0019 
.0028 
.0030 
.0006 
.0021 
.0039 
.0037 
.0022 
.0017 
.0028 
.0025 
.0031 
.0018 
.0016 
.0014 
.0030 
.0036 
.0022 
.0029 
.0050 
.0030 
.0049 
.0048 
.0010 

Per  cent. 
0.00074 
.00095 
. 00105 
.00091 
.00095 
.00095 
.00130 
.00134 
.00131 
.00115 
.00132 
.00128 
.00058 
.00066 
.00070 
.00053 
.00061 
.00062 
.00049 
.00066 
.00062 
.00087 
.00088 
.00086 

.139 

.139 

.143 

.140 

.0014 
.0052 

.138 

.0021 
.0007 
.0003 

.139 

.0034 
.0007 
.0024 
.0068 

.141 

The  method  of  Gray  in  a  slightly  modified  form  was  used  in  the 
summer  of  1908  and  the  spring  of  1909.  The  results  obtained  were, 
in  general,  similar  to  those  obtained  by  Gray.  They  seemed  to 
indicate  that  slow  proteolysis  was  taking  place. 

After  using  the  method  for  a  short  time  several  improvements 
suggested  themselves.  It  will  be  noticed  in  Table  1  that  the  largest 
amount  of  nitrogen  estimated  in  a  ferric-chlorid  filtrate  (column 
headed  "total  soluble  nitrogen")  was  equivalent  to  0.009  per 
cent  of  nitrogen  in  the  butter,  or  to  6.3  cubic  centimeters  N/10 
nitrogen.  This  is  not  a  large  amount.  The  largest  difference  between 
total  soluble  nitrogen  before  and  after  storage  in  Table  1  is  that  for 
sample  10.321,  and  is  equivalent  to  not  quite  4  cubic  centimeters 
N/10  nitrogen.  It  seemed  desirable  so  to  change  the  method  as  to 
increase  the  amounts  of  nitrogen  actually  estimated.  Whether 
proteolysis  did  or  did  not  take  place  would  then  be  decided  with 
the  aid  of  figures  that  are  not  so  small  that  the  unavoidable  errors 
in  such  work  are  comparatively  large.  Filtration  was  so  slow  that 
evaporation  undoubtedly  took  place  to  an  unnecessarily  large  extent. 
The  analyst  could  not  be  certain  that  100  cubic  centimeters  of 
a  ferric-chlorid  filtrate  obtained  after  storage  corresponded  to  ex- 
actly the  same  weight  of  butter  as  an  equal  volume  of  filtrate 
obtained  before  storage.  Either  filtration  must  be  so  rapid  that 
evaporation  may  be  disregarded  because  of  its  slight  extent,  or  if 
filtration  must   be  slow  the  volumes  of  filtrates  should  be  care- 


14  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

fully  measured,  so  that  the  weight  of  butter  corresponding  to  any 
volume  of  filtrate  can  be  definitely  ascertained. 

Objections  to  ferric  chlorid  and  tannic  acid  as  protein  precipitants. — 
Ferric  chlorid  as  a  protein  precipitant  was  not  wholly  desirable 
because,  as  here  used,  it  precipitated  not  alone  the  casein  and  other 
undigested  proteins,  but  their  immediate  digestion  products  down 
to  the  peptone  stage.  The  " f erric-chlorid  filtrate"  probably  does 
not  contain  proteoses.  The  use  of  the- term  "total  soluble  nitrogen" 
in  this  connection  will  lead  to  no  confusion  if  it  be  borne  in  mind  that 
it  means  nitrogen  not  precipitated  by  ferric  chlorid.  The  figures 
for  "proteose  and  peptone  nitrogen"  in  Table  1  probably  represent 
only  some  of  the  simpler  peptones.  The  amount  of  nitrogen  left  in 
the  f erric-chlorid  filtrate  is  small,  at  least,  when  used  for  the  separation 
of  the  different  forms  of  nitrogen  in  butter-curd  solution. 

Tannic  acid  as  a  protein  precipitant  is  perhaps  still  more  objection- 
able than  ferric  chlorid.  It  is  well  known  that  the  precipitation 
limits  of  tannic  acid  may  be  varied  by  the  presence  of  salts,  etc. 
Two  results  obtained  with  the  aid  of  tannic  acids  are  comparable 
only  when  the  precipitant  has  been  used  in  both  cases  under  condi- 
tions that  are  exactly  alike  as  regards  the  concentration  of  sodium 
chlorid,  the  amount  of  protein  to  be  precipitated,  the  precipitating 
power  of  the  samples  of  tannic  acid  used,  etc.1  The  greatest  care 
must  be  taken  in  the  use  of  the  reagent  to  insure  absolute  uniformity 
in  procedure.  This  is  shown  by  the  voluminous  literature  on  the 
use  of  this  reagent,  in  which  the  numerous  difficulties  and  necessary 
modifications  are  pointed  out. 

In  addition  to  the  difficulties  just  mentioned  are  those  resulting 
from  differences  of  opinion  among  investigators  as  to  the  best 
method  of  using  the  reagent.  Van  Slyke  and  Hart,2  in  their  deter- 
minations of  peptones  in  cheese  are  extremely  careful  to  avoid  an 
excess  of  tannic  acid,  probably  because  of  the  alleged  solubility  of 
the  precipitate  in  excess  of  the  precipitant. 

According  to  Bigelow  and  Cook  3 — 

*  *  *  a  considerable  excess  of  tannin  may  be  employed  without  any  tendency  of 
the  reagent  to  dissolve  the  precipitate  formed  in  excess.    *    *    * 

Gray  used  tannic  acid  as  directed  by  Van  Slyke  and  Hart. 

It  is  not  surprising  that  certain  workers  should  advocate  the  dis- 
continuance of  the  use  of  tannic  acid  as  a  reagent  for  the  determina- 
tion of  amino  acid  nitrogen.4 

1  Bigelow,  W.  D.,  and  Cook,  F.  C  The  separation  of  proteoses  and  peptones  from  the  simpler  amino 
bodies.    Journal  of  the  American  Chemical  Society,  vol.  28,  no.  10,  pp.  1485-1499.    Easton,  October,  1906. 

iLoc.  dt. 

»  Loc.  cit.,  p.  1493. 

<  Proceedings  of  the  Twenty-sixth  annual  convention  of  the  Association  of  Official  Agricultural  Chemists, 
United  States  Department  of  Agriculture,  Bureau  of  Chemistry,  Bulletin  132.  Washington,  1910.  See 
p.  156. 


PROTEOLYSIS  IN   BUTTER.  15 

The  results  obtained  by  Gray  for  amino  nitrogen  in  Table  1  are  not 
concordant,  probably  due  to  difficulties  inherent  in  the  use  of  tannic 
acid.  Our  own  results  are  likewise  difficult  to  interpret.  In  a  certain 
series  of  analyses  (butter  No.  13.5)  less  amino  nitrogen  was  founi  in 
the  butter  after  storage  than  before.  The  figures  are  not  given  here, 
as  they  are  essentially  similar  to  those  of  Gray. 

After  obtaining  a  considerable  number  of  results  on  the  distribution 
of  nitrogen  in  butter  before  and  after  storage,  with  the  aid  of  ferric 
chlorid  and  tannic  acid,  we  were  unable  to  conclude  that  the  results 
proved  anything.  It  seemed  more  and  more  desirable  to  perfect  a 
method  that  would  permit  the  precipitation  of  the  casein,  then  the 
estimation  of  proteoses  by  zinc  sulfate,  peptones  by  tannic  acid,  and 
ammonia  by  any  of  the  methods  that  did  not  give  too  high  results. 

In  order  to  obtain  a  filtrate  containing  sufficient  nitrogen  for 
analytic  work,  the  curd  solution  can  not  be  diluted  very  much.  In 
the  undiluted  condition  it  is  a  thick,  viscous  suspension  of  casein 
containing  a  variable  amount  of  fat  to  which  acetic  acid  may  be  added 
without  any  apparent  effect.  No  flocculation  can  be  observed.  The 
mixture  would  filter  extremely  slowly.  We  made  many  attempts  to 
find  out  why  filtration  was  so  slow.  At  first  it  was  thought  that  fat 
particles  clogged  the  filter  paper.  The  first  attempts  were  centered 
on  the  more  thorough  separation  of  the  butter  fat  from  the  remainder 
of  the  butter,  which  in  this  paper  is  referred  to  as  butter  curd  solution. 
It  is  obvious  that  unless  the  same  amount  of  fat  is  removed  in  com- 
parative analyses  of  butter  before  and  after  storage,  an  error  will 
be  introduced  because  the  fat  remains  in  the  precipitate,  giving  a 
smaller  volume  of  more  concentrated  filtrate.  The  error  from  this 
source  is  probably  much  larger  than  anyone  might  suppose.  The 
amount  of  fat  present  in  the  butter  curd  solution  used  for  analytical 
purposes  should  be  estimated  so  that  corrections  can  be  made  if 
necessary. 

The  butter  fat  may  be  separated  from  the  remainder  of  the  butter 
in  more  than  one  way.  But  obviously,  when  the  curd  solution  and 
not  the  fat  is  wanted  for  quantitative  work,  the  separation  must  con- 
sist of  something  more  than  a  mere  decantation  of  the  melted  fat. 
The  butter  fat  and  curd  are  so  thoroughly  mixed  in  the  butter  that 
when  the  butter  is  melted  and  allowed  to  stand,  the  separation  between 
fat  and  curd  solution  is  not  complete.  Most  of  the  fat  can  be  decanted, 
but  very  appreciable  amounts  still  remain  in  the  curd.  Although 
Gray  attempted  to  wash  out  the  fat  with  the  aid  of  gasoline,  it  is  cer- 
tain that  very  much  fat  was  still  present  in  the  material  used  for 
analysis.  We  tried  to  remove  the  fat  with  ether,  but  without  success. 
The  fat  particles  are  embedded  in  curd  and  in  this  condition  ether 
can  not  reach  them.  Besides,  the  ether  could  only  be  poured  on  top 
of  the  curd  solution.     Thorough  mixing  was  inadvisable  because  of 


16  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

the  possibility  of  forming  emulsions  that  could  not  be  separated.  The 
use  of  ether  was  soon  discontinued  because  of  the  possible  dehydrating 
action  of  the  ether  upon  the  protein  material  and  the  subsequent 
obscuring  of  the  results  for  nitrogen.  The  complete  separation  of  fat 
from  curd  solution  was  then  abandoned  with  the  intention  of  esti- 
mating the  amounts  of  fat  present  in  portions  used  for  analysis.  In 
their  work  on  storage  butter,  Rahn,  Brown,  and  Smith  1  did  not  record 
a  quantitative  separation  of  butter  fat  from  curd  solution.  Instead, 
they  proceeded  as  follows : 

Into  a  weighed  2-quart  [fruit]  jar  about  500  grams  of  butter  were  poured  and  weighed 
to  the  0.5  of  a  gram.  Then  2  grams  of  hot  water  (70°  C.  to  75°  C.)  for  every  gram  of 
butter  was  poured  into  the  jar  and  the  water  and  butter  stirred  occasionally  for  about 
an  hour.  The  cover  to  which  an  arrangement  for  letting  in  air  and  drawing  off  water 
had  been  attached  was  put  on  and  the  jar  inverted.  After  15  or  20  minutes  the 
water  which  had  been  separated  from  the  fat  was  drawn  off.  Aliquot  parts  were 
taken  for  analysis. 

One  objection  to  such  a  procedure  lies  in  the  fact  that  the  water  so 
separated  from  the  fat  contains  too  little  nitrogen.  The  analytic  data 
of  Rahn,  Brown,  and  Smith  on  nitrogen  partition  in  butter  are  open 
to  the  same  criticism  as  are  Gray's  data  in  Table  1.  Their  results 
differ  little  from  those  of  Gray.  Their  work  did  not  assist  in  explain- 
ing why  acetic  acid  will  flocculate  casein  in  milk,  but  not  in  butter 
curd  solution.  This  problem  had  been  evaded  by  practically  all 
who  studied  butter  chemistry. 

In  a  recent  investigation  on  the  influence  of  preservatives  on  the 
keeping  qualities  and  composition  of  butter  and  oleomargarin, 
Fischer  and  Gruenert 2  used  the  methods  of  Rahn,  Brown,  and  Smith 
in  their  studies  on  proteolytic  changes.  They  state  that  the  addition 
of  3  per  cent  of  salt  to  butter  greatly  retards,  but  does  not  entirely 
prevent,  proteolytic  and  other  changes  in  butter  stored  in  a  cool  cellar. 

NEW    METHOD   FOR    DETECTING    PROTEOLYSIS    IN    BUTTER. 

The  influence  of  sodium  chlorid  on  the  precipitability  of  casein  by 
acetic  acid. — Butter  curd  solution  differs  from  milk  in  many  respects; 
one  of  them  is  that  butter  curd  solution  may  contain  sodium  chlorid 
in  amounts  ranging  from  nothing  up  to  saturation  (over  30  per  cent), 
depending  upon  the  moisture  and  salt  content  of  the  butter.  Per- 
haps the  presence  of  the  salt  prevented  flocculation  of  casein. 

During  a  previous  investigation  on  the  temperature  of  pasteuriza- 
tion for  butter  making 3  the  following  method  of  precipitating  casein 

1  Loc.  cit.,  p.  14. 

5  Fischer,  K.,  and  Gruenert,  O.  tlber  den  einfluss  einiger  Konservierungsmittel  auf  Haltbarkeit  und 
Zusammensetzung  von  Butter  und  Margarine.  Zeitschrift  fur  Untersuchung  der  Nahrungs-  und  Genuss- 
mittel,  vol.  22,  no.  10,  pp.  553-582,  Berlin,  Nov.  15, 1911. 

'  Rogers,  L.  A.,  Berg,  W.  N.,and  Davis,  Brooke  J.  The  temperature  of  pasteurization  for  buttermaking. 
United  States  Department  of  Agriculture,  Bureau  of  Animal  Industry,  Circular  189,  Washington,  1912. 
See  p.  317. 


PROTEOLYSIS  IN  BUTTER.  17 

from  buttermilk,  milk,  or  skim  milk  was  used.  The  object  was  to 
obtain  a  filtrate  that  was  as  concentrated  in  nitrogen  as  possible,  and 
from  which  the  casein  had  been  quantitatively  separated : 

Transfer  200  cubic  centimeters  of  buttermilk  to  a  500  cubic  centimeter  volumetric 
flask.  Add  distilled  water  to  about  450  cubic  centimeters.  Add  one-fifth  normal 
acetic  acid  (1.2  per  cent)  slowly  until  the  casein  separates  completely  in  large  flocculi 
leaving  the  supernatant  liquid  practically  water-clear.  In  practically  every  case 
44  cubic  centimeters  of  N/5  acetic  acid  was  used  and  found  sufficient  for  the  purpose. 
After  diluting  to  the  mark  and  filtering,  nitrogen  determinations  were  made  on  the 

filtrate. 

■  -  -  - 

The  questions  to  be  studied  now  were:  Would  the  presence  of  salt 
in  milk  prevent  the  flocculation  of  the  casein  and  the  subsequent 
attempt  at  filtration  ? 

In  a  sample  of  skim  milk  from  which  the  casein  can  be  easily  floc- 
culated and  filtered  would  the  presence  of  added  fat  interfere  with 
filtration  ? 

Results  were  almost  immediately  obtained  which  threw  a  great 
deal  of  light  on  the  difficulty.     The  following  experiment  is  typical: 

Two  hundred  cubic  centimeters  of  buttermilk  obtained  from  a  churning  of  pas- 
teurized cream  were  transferred  to  a  500  cubic  centimeter  volumetric  flask.  Water 
was  added  to  about  400  cubic  centimeters.  On  slowly  adding  85  cubic  centimeters 
N/10  acetic  acid  (0.6  per  cent)  the  casein  was  completely  flocculated.  To  a  second 
500  cubic  centimeter  volumetric  flask  200  cubic  centimeters  of  the  same  sample  of 
buttermilk  was  transferred.  Thirty-six  grams  of  sodium  chlorid  were  added.  This 
concentration  of  approximately  18  per  cent  is  the  concentration  of  salt  in  butter  curd 
solution  when  the  butter  contains  16  per  cent  moisture  and  3  per  cent  salt.  Water 
was  added,  as  before,  to  about  400  cubic  centimeters.  The  addition  of  85  cubic  centi- 
meters N/10  acetic  acid  as  before  did  not  flocculate  the  casein. 

This  experiment  showed  that  the  presence  of  salt  very  materially 
affected  the  precipitability  of  the  casein.  This  was  clearly  a  case 
where  the  physical  condition  of  a  colloid  was  so  altered  by  the  pres- 
ence of  large  amounts  of  electrolytes  that  it  did  not  react  toward  a 
precipitant  as  it  usually  does.  The  acetic  acid  added  certainly  was 
not  in  excess.  The  slow  addition  of  the  acid  without  at  any  time 
resulting  in  even  a  partial  precipitation  of  casein  indicated  that  more 
acid  was  required.  After  adding  40  cubic  centimeters  more  of  N/10 
acetic  acid,  without  any  signs  of  flocculation,  10  per  cent  acetic  acid 
(5/3  normal),  was  carefully  added  instead  of  the  weaker  N/10  acid. 
After  adding  11  cubic  centimeters  of  10  per  cent  acetic  acid,  the 
casein  was  completely  flocculated,  and  the  mixture  could  be  filtered 
rapidly,  yielding  a  perfectly  clear  filtrate. 

In  the  presence  of  salt  300  cubic  centimeters  N/10  acetic  acid  were 
required  for  the  precipitation  of  casein,  which  in  the  absence  of  salt 
would  have  been  precipitated  by  85  cubic  centimeters  of  N/10  acetic 
acid.  It  is  probable  that  observations  of  a  somewhat  similar  nature 
were  simultaneously  made  by  other  investigators,  as  is  evident  from 
66711°— Bull.  162—13 3 


18  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

the  following  quotation  from  a  paper  by  Schryver  '  which  appeared  a 
few  months  after  the  above-described  experiment  was  made : 

During  the  course  of  some  investigations  on  the  action  of  formaldehyde  on  the  pro- 
teins, the  observation  was  made  that  this  aldehyde,  when  added  to  an  aqueous  solution 
of  Witte's  peptone,  produces  a  precipitate,  and  that  the  reaction  could  be  either 
partially  or  completely  inhibited  by  the  presence  of  neutral  salts.  This  phenomenon 
was  also  noticed  some  years  ago  by  T.  Sollman  (American  Journal  of  Physiology,  1902, 
vol.  7,  p.  220).    *    *    * 

Besides  being  extremely  interesting  theoretically,  the  observation 
just  made  on  the  effect  of  salt  on  the  precipitation  of  casein  was  of 
interest  practically  because  of  its  possible  application  to  the  separa- 
tion of  casein  from  butter  curd  solution.  The  theoretical  side  of  the 
phenomenon  is  discussed  by  Schryver  in  the  above-mentioned  paper. 

Method  for  the  estimation  of  water-soluble  nitrogen  in  butter. — After 
numerous  experiments  the  following  procedure  was  adopted  for  the 
separation  of  the  fat  from  the  butter  curd  solution  to  be  used  for 
nitrogen  determinations.  As  the  method  was  new,  many  precautions 
were  taken,  some  of  which  were  later  found  to  be  unnecessary.  This 
method  of  separating  fat  and  casein  from  butter  yields  a  filtrate  that  is 
well  adapted,  not  alone  to  a  study  of  the  nitrogenous  constituents  of  the 
filtrate,  but  to  many  other  purposes  as  well.  The  filtrate,  for  example, 
contains  the  peroxidase  which  is  practically  always  present  in  butter. 

To  two  or  three  large  beakers,  capacity  2  to  2£  liters,  transfer  5  to  6 
kilograms  of  the  sample  of  butter  to  be  studied.  Two  samples  (one 
from  raw,  one  from  pasteurized  cream,  for  example)  may  be  worked 
with  at  one  time.  The  larger  the  amount  of  butter  taken  the  better. 
Place  the  beakers,  properly  marked,  in  a  hot-air  bath  maintained  at 
about  45°  C.  If  the  butter  to  be  stored  is  packed  in  cans,  five  or  six 
2-pound  cans  are  placed  in  the  hot-air  bath  to  be  melted.  In  the 
work  here  described  the  cans  were  taken  out  of  the  ice  box  late  in  the 
afternoon,  allowed  to  warm  up  during  the  night  at  room  temperature, 
and  placed  the  next  morning  in  the  air  bath,  the  temperature  of 
which  varied  usually  2°  above  and  below  45°  C.  The  temperature 
should  not  be  permitted  to  rise  much  beyond  45°  C,  because  of  the 
danger  of  coagulating  some  of  the  protein  present.  The  high  tem- 
perature at  which  Gray  and  Kahn,  Brown  and  Smith  (see  pp.  11,  16) 
melted  their  samples  of  butter  undoubtedly  removed,  by  coagula- 
tion, comparatively  large  amounts  of  nitrogen  from  their  filtrates. 

At  this  temperature  from  six  to  eight  hours  will  be  required  for  the 
complete  melting  of  the  butter  and  the  settling  of  the  curd  solution. 
Stirring  does  little  good.  As  fast  as  the  butter  fat  forms  a  clear 
layer  on  top  of  the  butter  it  may  be  poured  off,  care  being  taken  that 
none  of  the  curd  solutionis  lost  at  any  time.  The  loss  of  a  few  small 
particles  of  solid  protein  is  not  material.     This  long  heating  at  a  tern- 

i  Schryver,  S.  B.  Some  investigations  dealing  with  the  state  of  aggregation  of  matter.  Proceedings  of 
the  Royal  Society,  London,  series  B,  vol.  83,  no.  B562,  pp.  96-123.    London,  Dec.  19, 1910.    See  p.  96. 


PROTEOLYSIS   IN   BUTTER.  19 

perature  that  is  perhaps  best  for  proteolytic  action  is  an  objection  to 
the  method.  It  will  be  apparent,  however,  that  the  error  introduced 
in  this  way  is  inappreciable.     (See  p.  20.) 

The  melted  butter  fat  is  decanted  until  no  more  can  be  so  removed 
without  danger  of  losing  some  of  the  curd  solution.  With  the  aid  of  a 
100  cubic  centimeter  pipette  having  its  lower  tip  cut  off  to  permit 
more  rapid  flow  of  viscous  materials,  remove  the  curd  solution  from 
the  bottom  of  the  beaker  or  can  and  transfer  it  to  a  dry,  clean,  500 
cubic  centimeter  volumetric  flask.  Considerable  fat  will,  of  course 
be  mixed  with  the  curd  solution,  but  by  taking  only  part  from  the 
bottom  of  each  vessel  sufficient  curd  solution  will  be  obtained  from 
5  kilograms  of  butter  to  fill  two  500  cubic  centimeter  flasks.  Let 
stand  till  the  next  morning  at  room  temperature.  With  the  aid  of  a 
rapid-flow  100  cubic  centimeter  pipette,  remove  from  the  bottom  of 
each  of  these  flasks  between  200  and  250  cubic  centimeters  of  curd 
solution  and  transfer  this  to  a  clean,  dry,  500  cubic  centimeter  volu- 
metric flask.  From  the  original  5  kilograms  of  butter  there  have  now 
been  separated  not  quite  500  cubic  centimeters  of  curd  solution  con- 
taining approximately  10  per  cent  of  fat.  The  rest  of  the  curd  solu- 
tion containing  much  larger  proportions  of  fat  may  be  rejected. 

In  a  separate  portion  of  the  original  sample  of  butter,  determine 
moisture,  curd,  and  salt.  For  this  purpose  the  methods  described  in 
Bulletin  107  (revised  edition),  Bureau  of  Chemistry,  page  123,  were 
used.  From  these  figures  the  weight  of  butter  corresponding  to  a 
given  volume  of  curd  solution  may  be  calculated  if  desired,  but  for 
the  present  work  such  calculation  was  not  necessary. 

The  curd  solution  should  be  well  mixed,  and  then  with  the  aid  of  a 
rapid-flow  pipette  a  portion  is  transferred  to  a  pycnometer  (50  cubic 
centimeters  capacity)  and  the  weight  ascertained.  The  object  of  this 
determination  is  to  make  certain  that  the  curd  solution  used  for 
analysis  before  and  after  storage  of  butter  is  practically  the  same  so 
far  as  the  density  of  the  material  is  concerned.  It  is  obvious  that  if 
the  proportion  of  fat  and  salt  solution  differs  very  much  in  two  sam- 
ples of  curd  solution  obtained  from  the  same  lot  of  butter  before  and 
after  storage  the  specific  gravity  will  be  different.  This  determina- 
tion, when  repeated  on  the  same  portion  of  curd  solution,  will  show 
that  it  is  possible  to  withdraw  samples  of  uniform  composition  from 
the  flask  if  care  be  taken  to  mix  the  contents  well  and  to  withdraw 
the  sample  rapidly  before  the  fat  rises  to  any  appreciable  extent. 

Portions  of  this  curd  solution  may  now  be  withdrawn  for  nitrogen 
determinations.  To  several  clean,  dry,  500  cubic  centimeter  volu- 
metric flasks  transfer  100  cubic  centimeter  portions  of  curd  solution, 
using  a  rapid-flow  pipette  and  sampling  quickly.  This  is  the  amount 
found  most  convenient  when  acetic  acid  is  to  be  used  as  a  precipitant. 
When  other  precipitants,  such  as  ferric  chlorid,  are  to  be  used,  a  larger 
volume  may  be  taken. 


20  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

In  our  work  we  generally  made  three  and  sometimes  four  deter- 
minations on  a  single  sample  of  butter,  although  two  are  perhaps 
sufficient.  Each  100  cubic  centimeter  portion  was  used  for  one 
determination,  so  that  as  many  such  portions  are  to  be  pipetted  into 
500  cubic  centimeter  flasks  as  determinations  are  wanted.  (See 
Table  2.)  In  the  remainder  of  the  curd  solution  we  determined  fat 
by  removing  10  cubic  centimeter  portions,  transferring  to  absorbent 
paper,  drying  at  the  temperature  of  boiling  water,  and  extracting  the 
fat  in  a  Soxhlet  apparatus,  as  usual.  These  figures  for  fat  are  for 
the  purpose  of  control  only,  and  indicate  how  much  of  the  volume 
of  the  portion  of  curd  solution  is  taken  up  by  fat. 

The  undiluted  curd  solution  may  be  allowed  to  remain  in  the 
stoppered  flasks  for  24  or  48  hours  at  room  temperature  without  any 
apparent  change  in  the  results.  In  several  cases  even  a  week's 
standing  was  without  effect.  The  concentration  of  salt  is  usually 
high  enough  to  make  the  addition  of  other  preservatives  unnecessary. 
Under  these  conditions  the  proteolytic  enzym  present,  galactase,  is 
not  very  active;  at  least,  on  standing  several  days  practically  the 
same  results  are  obtained  as  on  freshly  prepared  curd  solution. 

The  curd  solution  is  diluted  to  about  450  cubic  centimeters  with 
distilled  water,  mixed  well  and  then  10  per  cent  acetic  acid  is  added 
slowly,  with  constant  mixing  of  the  contents  of  the  flask.  It  is 
obvious  that  sufficient  acetic  acid  must  be  added  to  completely  pre- 
cipitate the  casein  in  the  flocculent  condition.  This  will  occur 
generally  when  18  to  22  cubic  centimeters  have  been  added.  The 
curd  solutions  from  pasteurized  cream  butter  generally  require  the 
larger  amount  for  flocculation.  In  Table  2  the  amounts  of  acetic 
acid  used  are  indicated.  It  seems  that  the  more  fat  present  the  more 
slowly  does  the  casein  flocculate.  On  standing  about  15  minutes  the 
casein  will  be  seen  to  have  flocculated.  The  amount  of  acetic  acid 
added  should  be  recorded  so  that  that  same  amount  can  be  used  after 
storage.  In  fact,  all  data  should  be  recorded  that  might  be  necessary 
for  the  purpose  of  duplicating  the  determination  exactly  after  storage. 
Dilute  with  distilled  water  to  the  mark  and  filter  on  a  32  centimeter 
folded  filter  (S.  &  S.  No.  588  or  595)  into  a  clean,  dry,  500  cubic 
centimeter  volumetric  flask.  The  curd  solutions  should  not  be 
allowed  to  stand  long  after  the  addition  of  water.  The  filter  stands, 
(clean  runnels,  and  flasks  were  in  readiness  before  the  addition  of  water 
«,nd  acid  to  the  curd  solutions,  so  that  filtration  was  begun  within  a 
very  few  minutes.  The  entire  contents  of  the  flask  were  transferred 
to  the  filter  paper.  If  the  filtrate  at  first  was  cloudy,  it  was  returned 
to  the  filter  as  often  as  necessary.  Usually  the  first  portions  of 
filtrate,  about  50  cubic  centimeters  each,  were  returned  to  the  filter 
two  or  three  times.  In  no  case  was  a  filtrate  used  for  nitrogen  deter- 
minations that  was  so  cloudy  as  to  indicate  the  presence  of  unpre- 
cipitated  casein.     Usually  the  filtration  was  begun  in  the  afternoon 


PROTEOLYSIS  IN   BUTTER.  21 

and  allowed  to  go  on  till  the  next  morning.  The  funnel  was  covered 
with  a  well-fitting  watch  glass  to  minimize  evaporation. 

On  several  occasions  the  precipitate  on  the  filter  paper  was  exam- 
ined at  the  end  of  the  filtration  for  peptonizing  bacteria.  Such  small 
numbers  were  found  that  their  effect  was  inappreciable.  It  is 
obvious  that  the  digestion  of  protein  on  the  filter  paper  by  bacteria 
or  their  enzymes  might  vitiate  the  results.  We  have  no  reason  to 
believe  that  in  any  of  the  results  appreciable  errors  were  introduced 
in  this  way. 

The  amount  of  acetic  acid  was  varied  a  little  when  precipitating  the 
casein  from  different  portions  of  the  same  curd  solution  in  order  to 
find  out  whether,  under  the  conditions  of  the  work,  small  variations 
in  the  amount  of  acetic  acid  would  give  rise  to  undesirably  large 
variations  in  the  results.  Of  course,  if  insufficient  acetic  acid  is 
added  all  of  the  casein  will  not  be  precipitated  and  the  mixture  will 
filter  so  very  slowly  that  that  alone  will  indicate  incomplete  pre- 
cipitation. The  more  completely  the  casein  is  precipitated  the  more 
rapid  is  the  filtration.  Slight  excesses  of  acetic  acid  apparently  have 
an  inappreciably  small  solvent  action  on  the  precipitate.  It  is  true 
that  in  precipitating  casein  from  diluted  milk  an  excess  of  even  very 
dilute  acetic  acid  is  undesirable.  In  the  presence  of  the  sodium 
chlorid,  however,  conditions  are  so  altered  that  the  solvent  action  of 
the  acetic  acid  is  apparently  very  much  diminished.  In  case  of 
doubt,  more  rather  than  less  acetic  acid  was  used. 

The  clear,  slightly  opalescent  filtrate  may  be  tested  for  complete- 
ness of  precipitation  by  the  addition  of  more  acid  or  of  alkali.  In  no 
case  did  the  addition  of  small  amounts  of  acid  or  of  alkali  (N/10)  to  the 
filtrate  result  in  the  precipitation  of  protein.  If  sufficient  alkali  was 
added  to  make  the  filtrate  alkaline  to  phenolphthalein,  a  precipitate 
was  obtained  which  was  probably  calcium  phosphate  containing 
adsorbed  protein.  In  appearance  it  resembled  some  protein  pre- 
cipitates. The  appearance  of  such  a  precipitate  in  an  alkaline 
filtrate  may,  of  course,  be  disregarded.  Another  way  to  test  for 
completeness  of  precipitation  is  to  use  slightly  different  amounts  of 
the  precipitant.  The  results  tabulated  in  Table  2  show  that  the 
amounts  of  acetic  acid  used  were  sufficient  and  that  slight  variations 
in  the  strength  of  the  acid  made  no  difference  in  the  results.  For  the 
sake  of  certainty,  the  10  per  cent  acetic  acid  solutions  were  titrated 
against  standard  alkali  before  being  used  as  precipitants.  This  is 
especially  desirable  where  the  first  determination  is  made  in  one 
laboratory  and  the  second  after  cold  storage  in  another. 

In  a  few  instances  ferric  chlorid  was  used  as  a  precipitant  for  the 
purpose  of  comparing  the  results  with  those  obtained  with  acetic  acid. 

Since  evaporation  can  not  be  altogether  prevented  during  the  long 
filtration,  it  is  necessary  to  be  certain  that  equal  volumes  of  filtrates 
obtained  from  the  same  lot  of  butter  before  and  after  storage  corre- 


22  CHANGE   IN   FLAVOR   OP   STORAGE  BUTTER. 

spond  to  exactly  equal  weights  of  butter,  or,  if  through  any  con- 
siderable difference  in  evaporation  the  two  filtrates  are  unequally 
concentrated,  the  difference  in  concentration  must  be  ascertainable. 

After  filtration  is  nearly  complete — that  is,  after  obtaining  a  little 
over  400  cubic  centimeters  of  filtrate — its  specific  gravity  is  determined. 
A  50  cubic  centimeter  pycnometer  was  used.  The  same  pycnometer 
filled  a  second  time  with  some  of  the  same  filtrate  will  differ  from  its 
first  weight  by  only  1  milligram.  A  little  calculation  will  show  that 
before  the  nitrogen  content  of  the  filtrate  can  be  appreciably  varied 
through  evaporation,  the  specific  gravity  will  be  varied  so  much  more 
that  its  detection  will  be  easy  and  require  no  fine  weighings  of  the 
pycnometer.  The  pycnometer  full  of  filtrate  was  always  quickly 
.dried  and  weighed,  and  the  weight  recorded.  The  weight  of  a  known 
volume  of  the  clear  filtrate  is  the  best  of  the  control  figures,  and 
together  with  the  others  should  in  every  case  show  whether  or  not 
two  filtrates  of  supposedly  equal  concentration  really  were  so.  The 
container  in  which  the  butter  is  stored  might  leak.  There  would 
result,  not  alone  a  loss  in  moisture,  but  in  salt  and  nitrogen  as  well. 
Or  if  the  container  did  not  altogether  prevent  evaporation  of  water 
and  a  subsequent  concentration  of  salt  and  nitrogen  resulted,  the 
pycnometer  weighings  will  probably  indicate  the  source  of  variation. 
When  100  cubic  centimeters  of  curd  solution  were  used,  the  filtrate 
contains  so  much  sodium  chlorid  that  considerable  variation  in 
specific  gravity  is  possible.  The  amount  of  curd  solution  used  corre- 
sponded to  nearly  700  grams  of  butter. 

After  weighing  the  pycnometer  full  of  the  clear  filtrate,  400  cubic 
centimeters  of  it  were  transferred  to  a  Kjeldahl  flask  and  total 
nitrogen  was  determined  in  the  usual  way.  The  remainder  of  the 
filtrate  was  measured  and  the  total  volume  of  filtrate  obtained  was 
recorded.  If  filtration  was  very  slow,  sometimes  less  than  400  cubic 
centimeters  were  used.  The  results  were  calculated  to  400  cubic 
centimeters.  The  weight  of  the  precipitate  and  inclosed  filtrate  was 
ascertained  to  the  nearest  gram  on  a  torsion  balance,  and  recorded. 
The  400  cubic  centimeters  of  filtrate  actually  kjeldahled  and  titrated 
corresponded  to  560  grams  of  butter.  Rahn,  Brown,  and  Smith  * 
determined  nitrogen  in  butter  not  precipitated  by  acetic  acid.  They 
washed  butter  with  water  in  the  proportion  of  1  gram  of  butter  to 
2  cubic  centimeters  wash  water,  transferred  100  cubic  centimeters 
of  these  washings  to  a  flask,  added  acetic  acid,  filtered  off  the  pre- 
cipitated protein,  and  determined  total  nitrogen  in  25  cubic  centimeters 
of  filtrate,  which  corresponded  to  not  quite  12£  grams  of  butter. 
Apparently  they  obtained  very  few  results  with  acetic  acid  and  did 
not  use  them  in  drawing  their  conclusions.  It  was  pointed  out 
before  (see  p.  10)  that  when  butter  curd  solution  is  diluted  with 
sufficient  water  it  may  be  treated  with  acetic  acid  as  if  it  were  so 

»  Loc.  cit.,  pp.  14-15. 


PBOTEOLYSIS  IN   BUTTEE.  23 

much  diluted  milk  and  the  casein  will  be  flocculated,  permitting  very 
satisfactory  filtration.  The  filtrate,  however,  contains  an  unde- 
sirably small  amount  of  nitrogen. 

The  figure  for  total  nitrogen  in  the  filtrate  taken  for  analysis  was 
multiplied  by  5/4,  and  the  result  recorded  as  the  number  of  cubic 
centimeters  of  N/5  nitrogen  in  100  cubic  centimeters  butter  curd  solu- 
tion. (See  Table  2.)  From  the  data  it  is  obvious  that  the  unavoid- 
able errors  of  ordinary  nitrogen  determinations  are  very  small  when 
compared  with  the  amount  of  nitrogen  determined. 

A  separation  of  the  various  forms  of  nitrogen  in  the  filtrate  from 
the  casein  precipitation  could  easily  have  been  made.  But  it  was 
considered  desirable  first  to  find  out  whether  this  filtrate  contained 
any  more  nitrogen  after  storage  than  before.  If  it  did,  indicating 
that  proteolysis  was  taking  place,  then  a  more  detailed  examination 
of  the  filtrate  would  have  been  made.  But  the  filtrates  differed  very 
little  in  their  total  nitrogen  content  before  and  after  storage,  indicating 
that  proteolytic  changes  were  not  taking  place  to  any  great  extent, 
and  other  lines  of  work  were  begun. 

The  above-described  method  for  the  estimation  of  water-soluble 
nitrogen  in  butter  was  used  in  the  summer  of  1910  and  in  the  spring 
of  1911.     (See  Table  2.) 

Description  of  samples. — Samples  Nos.  42  and  40  were  churned  from 
the  same  lot  of  sweet  cream,  half  of  which  was  churned  unpasteurized 
(butter  No.  42)  and  half  of  which  was  churned  after  pasteurization 
(butter  No.  40) .  Samples  52  and  50,  and  samples  65  and  62  were 
likewise  obtained  from  churnings  of  two  lots  of  sweet  cream,  part  of 
which  was  pasteurized,  part  of  which  was  not,  before  churning. 
(See  Table  2.)  The  expectation  was  that  if  galactase  is  active 
in  butter  during  cold  storage,  the  figures  for  water-soluble  nitrogen 
in  samples  42,  52,  and  65  would  become  larger,  for  in  these  sam- 
ples of  butter  from  unpasteurized  cream  the  conditions  for  pro- 
teolysis were  as  favorable  as  they  ordinarily  can  be.  No  great 
changes  were  expected  in  the  water-soluble  nitrogen  in  the  controls 
Nos.  40,  50,  and  62  because  at  the  temperature  of  pasteurization 
used,  75°  C.  (167°  F.)  in  a  "flash"  pasteurizer,  the  galactase  ordi- 
narily present  in  butter  is  strongly  inactivated  or  partly  destroyed. 

Samples  511  and  523  were  churned  from  the  same  lot  of  pasteurized 
cream.  Sample  No.  51 1  contained  a  proteolytic  enzym  preparation 
obtained  from  cultures  of  acid-forming  bacteria  which  also  liquefied 
gelatin.  Twelve  grams  of  dry  enzym  powder  were  worked  into 
about  30  pounds  of  butter  with  the  salt.  The  control  lot  of  butter 
No.  523  was  made  in  the  same  way,  except  that  a  similar  amount  of 
enzym  preparation  was  added  after  it  was  first  boiled  in  water. 
Similarly,  sample  No.  466  was  churned  from  pasteurized  cream  and 
contained  an  added  proteolytic  enzym  preparation,  while  its  con- 
trol, No.  478,  contained  an  equal  amount  of  the  enzym  that  had 


u 


CHANGE   IN   FLAVOR   OP   STORAGE  BUTTER. 


been  boiled  before  being  worked  into  the  butter.  The  object  of  study- 
ing these  samples  was  to  ascertain  whether  the  proteolytic  enzym 
secreted  by  bacteria  often  present  in  cream  can  digest  any  of  the 
butter  proteins,  under  storage  conditions. 

All  of  the  samples  in  Table  2  were  churned  in  the  experimental 
creamery  in  Albert  Lea,  Minn.,  in  the  summer  of  1910.  They 
were  stored  a  short  time  in  the  creamery  cooler  and  then  shipped 
to  cold  storage  at  10°  F.  (minus  12°  C.)  in  Chicago.  Naturally,  care 
was  taken  to  move  the  material  into  cold  storage  as  soon  after  churn- 
ing as  possible.  When  samples  were  shipped  from  Chicago  to  Wash- 
ington for  analysis,  they  were  removed  from  the  railroad  station  as 
soon  after  their  arrival  as  possible  and  placed  in  a  refrigerator  in  the 
laboratory  maintained  at  a  few  degrees  below  0°  C. 

At  appropriate  times  samples  of  the  butter  were  sent  to  competent 
judges  for  scoring.  The  scores  are  indicated  in  their  appropriate 
places  in  Table  2. 

Table  2. — Analytic  data  and  scores — Water-soluble  nitrogen  '  in  sweet-cream  butter  be/ore 
and  after  storage.    10°  F.  (-12°  C). 


N/5  water-soluble 

nitro- 

gen  in  100  c.  c. 

butter 

Butter 

scores. 

Volume 

Precipi- 

Butter 

curd  solution. 

of  butter 

tant, 

Treatment  of  cream. 

sample 

Age  of 

curd 
solution 
used  for 
analysis. 

10  per 

No. 

Before 

After 

Differ- 

sample. 

Before 

After 

cent 
acetic 
acid. 

storage. 

storage. 

ence. 

storage. 

storage. 

C.c. 

C.c. 

C.c. 

Days. 

C.c. 

C.c. 

f       32.8 

28.9 

-  3.9 

f           50 

7 

Unpasteurized 

42 

34.0 

28.7 

—  5.3 

■      265 

85 

85 

<           50 

7 

19.9 

14.0 

-  5.9 

100 

(2) 

30.3 

31.9 

1.6 

I         100 

10 

52 

28.9 

30.3 

1.4 

•     251 

87 

85 

\          100 

14 

28.7 

29.0 

0.3 

100 

16 

26.1 

21.2 

-  4.9 

i     loo 

16 

65 

25.1 

21.2 

-  3.9 

■      250 

90 

\          100 

20 

26.0 

21.2 

-  4.8 

100 

22 

23.4 

24.6 

1.2 

f           50 

7 

Pasteurized 

40 

23.4 
13.4 

23.6 
16.0 

0.2 

2.6 

•      265 

91 

90 

\           50 
1          100 

7 

(2) 

19.4 

20.7 

1.3 

(          100 

16 

50 

19.0 

19.0 

0.0 

251 

93 

91 

I         100 

18 

18.7 

19.4 

0.7 

100 

20 

22.8 

17.6 

-  5.2 

f         100 

16 

62 

22.3 

17.6 

-  4.7 

•     250 

92 

\         100 

20 

22.7 

16.8 

-  5.9 

100 

22 

Pasteurized.   Dry  pro- 

1 

31.8 

26.6 

-  5.2 

f         100 

18 

teolytic  enzym 

\      511 

32.6 

26.5 

-  6.1 

•     250 

92 

92 

i          100 

20 

added' 

J 

35.1 

27.1 

-  8.0 

100 

12 

<32.7 

72.9 

40.2 

1   p> 

1    (7) 

(6) 

466 

<86.1 

86.6 

0.5 

•      296 

92J 

93 

9 

U17.9 

84.1 

-33.8 

(') 

5 

Pateurized.       Heated 

1 

22.4 

23.5 

1.1 

f         100 

18 

bacterial  proteolytic 

\      523 

22.8 

23.5 

0.7 

■      250 

93 

93 

\          100 

20 

J 

23.6 
«22.0 

24.4 

0.8 

100 

1  8 

1    (7) 

12 

(2) 

478 

<23.9 
«75.0 

55.0 
68.7 

31.1 
-  6.3 

296 

93* 

93J 

(3) 

9 

<112.8 

72.3 

-40.5 

1  P) 

5 

1  Calculated  total  nitrogen  in  100 cc  butter  curd  solution  equivalent  to  nearly  700  grams  butter=-350ccN/5 
nitrogen. 
1  Eight  c.  c.  10  per  cent  ferric  chlorid  solution. 

3  Analytical  work,  June,  1911,  on  butter,  buttermilk,  etc.,  by  R.  P.  Norton. 

4  N/5  water-soluble  nitrogen  in  1 ,000  grams  butter. 

5  Equivalent  of  800  grams  of  butter. 

«  Ten  c.  c.  10  per  cent  ferric  chlorid  solution. 
i  Equivalent  of  400  grams  of  butter. 


PEOTEOLYSIS  IN   BUTTEE.  25 

Discussion  of  results,  Table  2. — In  general,  fresh  butter  made  from 
unpasteurized  cream  (No.  42,  for  example)  has  a  little  more  water 
soluble  nitrogen  than  the  corresponding  sample  (No.  40)  churned 
from  some  of  the  same  lot  of  cream  after  pasteurization.  Butter- 
milk from  raw-cream  butter  contains  more  water-soluble  nitrogen 
than  the  corresponding  sample  of  buttermilk  from  pasteurized 
cream.  (Compare  samples  13  and  14,  Table  3.)  In  sterilized  skim 
milk  the  soluble  nitrogen  content  is  still  lower.  (Compare  samples 
20,  22,  and  24  with  14, 16,  and  18,  Tables  3  and  4.)  These  differences 
between  pasteurized  and  unpasteurized  samples  are  very  likely  due 
to  the  partial  or  entire  coagulation  of  the  milk  albumin,  and  its 
removal  from  the  water-soluble  condition. 

The  coagulation  of  water-soluble  nitrogenous  substances  in  butter- 
milk due  to  high  pasteurizing  temperatures  was  shown  in  a  previous 
publication  from  the  Dairy  Division  laboratories.1 

It  is  believed  that  samples  42,  52,  and  65  contained  more  water- 
soluble  nitrogen  than  their  controls  Nos.  40,  50,  and  62,  because 
there  was  a  partial  precipitation  of  protein  during  the  pasteuriza- 
tion of  the  cream  from  which  the  latter  were  made  and  not  because 
the  galactase  undoubtedly  present  in  Nos.  42,  52,  and  65  was  active. 
If  it  were,  there  should  have  been  an  increase  in  the  amount  of 
water-soluble  nitrogen  after  storage.  In  so  far  as  there  was  none, 
it  is  inferred  that  the  activity  of  the  galactase  was  inhibited  by  the 
combined  effect  of  the  salt  and  cold  storage. 

The  differences  between  the  amounts  of  water-soluble  nitrogen  in 
the  different  samples  of  butter  before  and  after  storage  are  not  very 
large,  except  in  samples  466  and  478,  and  they  represent  in  all 
probability  the  unavoidable  error  in  such  work.  It  is  to  be  borne 
in  mind  that  the  first  analysis  was  made  in  Albert  Lea,  Minn.,  and 
the  second  on  a  different  lot  of  cans  in  Washington,  D.  C.  Under 
such  circumstances  the  differences  are  not  considered  large.  In 
samples  466  and  478  it  is  probable  that  the  figures  obtained  for 
nitrogen  are  erroneous. 

CONCLUSIONS. 

From  the  data  obtained  it  is  evident  that  proteolysis  did  not  take 
place  to  any  appreciable  extent  in  the  samples  studied.  Nor  was 
there  any  simple  or  obvious  relation  between  the  figures  for  nitrogen 
and  the  butter  scores. 

As  the  following  calculations  show,  the  method  for  detecting 
proteolytic  action  in  butter  is  quite  delicate  and  should  lead  to  the 
detection  of  proteolysis  were  it  appreciable.  In  a  'determination  of 
water-soluble  nitrogen  in  butter  100  cubic  centimeters  of  curd  solu- 
tion were  used.     This  is  equivalent  to  a  little  over  700  grams  of  butter 

1  Rogers,  L.  A.,  Berg,  W.  N.,  and  Davis,  Brooke  J.    Loc.  cit.,  p.  317. 
66711°— Bull.  162—13 4 


26  CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 

containing  13  per  cent  of  moisture.  A  nitrogen  determination  was 
made  in  400  cubic  centimeters  of  the  filtrate  from  the  acetic  acid 
precipitation,  equivalent  to  560  grams  of  butter.  The  butter  used 
contained  an  average  of  0.9  per  cent  of  curd.  The  700  grams  of 
butter  contained,  therefore,  700X0.009X0.1567  =  0.9872  grams  of 
nitrogen,  equivalent  to  352  cubic  centimeters  N/5  nitrogen. 

Suppose  that  during  the  cold-storage  period  5  per  cent  of  the  casein 
was  slightly  proteolyzed  and  became  water  soluble.  It  should  be 
borne  in  mind  that  the  method  of  detecting  proteolytic  action  here 
described  will  detect  it  in  its  first  stages.  In  this  respect  the  method 
possesses  undoubted  advantages  over  others  in  which  results  are 
obtained  for  variations  hi  amounts  of  proteoses,  amino  acids  or 
ammonia,  which  correspond  to  later  and  later  stages  in  the  digestive 
process.  It  is  here  supposed  that  the  first  step  in  the  digestion  of 
5  per  cent  of  the  casein  has  taken  place,  the  rest  of  the  protein 
remaining  unchanged.  Allowing  for  30  cubic  centimeters  N/5  nitro- 
gen already  present  in  the  100  cubic  centimeters  of  curd  solution 
as  water-soluble  nitrogen,  there  would  be  formed  by  the  proteolysis 
0.05  X  320  cc  =  16  cubic  centimeters  N/5  nitrogen  in  the  water  soluble 
form  in  addition  to  the  30  cubic  centimeters  originally  present.  The 
titrations  made  after  storage  would  then  show  16x4/5  or  12  cubic 
centimeters  more  of  N/5  nitrogen  than  before  storage.  From  the 
results  obtained  it  is  probable  that  this  increase,  had  it  taken  place, 
would  have  been  detected. 

A  protein,  such  as  casein,  can  of  course  undergo  more  than  one 
kind  of  chemical  change.  These  changes  may  be  hydrolytic,  oxida- 
tive, or  putrefactive.  It  is  obvious  that  the  methods  used  in  this 
work  would  detect  the  hydrolytic  change  only.  Some  work  on  the 
possibility  of  oxidative  changes  in  the  protein  in  butter  is  described 
on  page  64  et  seq. 

PROTEOLYSIS  IN  MILK. 

POSSIBLE    OBJECTION    TO    THE    NEW    METHOD    FOR    DETECTING    PRO- 
TEOLYSIS   IN    BUTTER. 

An  apparent  objection  to  the  method  of  studying  possible  pro- 
teolytic changes  in  butter  just  described  lies  in  the  fact  that  a 
long  time  (five  days)  may  elapse  between  the  beginning  and  end 
of  the  determinations  of  nitrogen,  during  which  time  the  galactase 
or  bacterial  proteolytic  enzyms  present  in  butter  may  be  active. 
The  results  would  not  represent  proteolysis  during  cold  storage  but 
proteolysis  during  the  determinations  of  nitrogen.  For  this  reason 
the  following  experiments  were  made. 

The  objects  of  these  were  two-fold :  First,  to  ascertain  by  a  method 
that  was  free  from  the  objections  to  the  method  for  butter  whether 


PKOTEOLYSIS  IN   MILK.  27 

galactase  could  digest  protein  in  an  18  per  cent  sodium  chlorid 
solution.  Second,  whether  the  sodium  chlorid  usually  present  in 
butter  curd  solutions  can  inhibit  the  proteolytic  action,  not  alone  of 
the  small  quantity  of  galactase  ordinarily  present  in  butter,  but  also 
the  action  of  larger  amounts  of  proteolytic  enzyms  that  might  find 
their  way  into  butter  in  any  one  of  several  ways,  as,  for  instance, 
the  proteolytic  bacteria  that  may  grow  in  the  milk  and  cream. 

THE  INHIBITING  EFFECT  OF  SODIUM  CHLORID  AND  COLD  STORAGE  UPON 
THE    ACTIVITY   OF   GALACTASE    IN   BUTTERMILK. 

Six  lots  of  buttermilk  of  about  eight  liters  each  were  obtained 
from  three  churnings  of  unpasteurized  sweet  cream  and  from  three 
churnings  of  pasteurized  sweet  cream.  In  Table  3,  page  29,  is  indi- 
cated the  number  of  the  lot  of  butter  corresponding  to  each  lot  of 
buttermilk.  Buttermilk  samples  13  and  14  were  obtained  from  the 
churning  of  one  lot  of  sweet  cream,  half  of  which  was  churned  un- 
pasteurized (butter  No.  42,  buttermilk  No.  13)  and  half  of  which 
was  pasteurized  before  churning  (butter  No.  40,  buttermilk  No.  14). 

To  each  liter  of  buttermilk  5  cubic  centimeters  of  chloroform  and 
180  grams  of  sodium  chlorid  were  added.  These  samples  were 
intended  to  represent  approximately  butter  minus  butterfat.  In 
such  material  a  study  of  possible  proteolytic  action  could  be  made 
in  a  comparatively  short  time,  since  no  time  is  necessary  for  the 
melting  of  the  butter  or  the  separation  of  the  fat. 

The  buttermilk  was  then  sealed  in  cans,  each  containing  600  cubic 
centimeters  of  the  sample.  These  cans,  which  were  also  used  for 
butter,  were  of  heavy  tin,  thoroughly  lacquered.  The  smaller  part 
of  these  samples  remained  in  the  creamery  cooler;  the  rest  were 
shipped  to  cold  storage  in  Chicago.  The  samples  were  not  removed 
from  the  cooler  for  analysis  until  it  was  certain  that  sufficient  time 
had  passed  to  permit  the  transportation,  by  refrigerator  freight,  of 
the  samples  from  Albert  Lea,  Minn.,  to  Chicago,  and  the  placing  of 
the  samples  in  the  cold-storage  rooms.  It  was  intended  that  the 
first  analysis  of  these  samples  should  show,  as  closely  as  possible, 
the  amount  of  water-soluble  nitrogen  present  in  the  material  as  it 
went  into  cold  storage,  and  not  as  it  left  the  churn.  It  is  highly 
probable  that  galactase,  even  in  the  presence  of  18  per  cent  sodium 
chlorid,  can  slowly  digest  protein  material,  if  the  temperature  is 
above  that  of  cold  storage  as  it  is  ordinarily  practiced.  It  seems 
reasonable  to  suppose  that  the  proteolysis  in  butter  observed  by 
Rahn,  Brown,  and  Smith  1  was  due,  in  part  at  least,  to  the  compara- 
tively high  temperatures  at  which  their  butter  was  stored.  But  in 
so  far  as   this  investigation  is  concerned   with  possible  chemical 

» Loc.  clt. 


28  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

changes  that  may  take  place  in  butter  while  in  cold  storage  and  not 
at  higher  temperatures,  the  times  at  which  analyses  were  made  were 
always  chosen  so  as  to  give  results  as  nearly  representative  of  the 
condition  of  the  material  immediately  before  and  after  cold  storage 
as  possible. 

Method  of  measuring  the  activity  of  galactase  in  buttermilk. — When 
it  was  reasonably  certain  that  the  other  samples  of  buttermilk  had 
reached  the  cold  storage,  samples  were  removed  from  the  creamery 
cooler  and  water-soluble  nitrogen  was  determined  in  them  by  prac- 
tically the  same  method  as  was  used  for  butter.  The  buttermilk 
was  treated  as  if  it  were  so  much  butter  curd  solution  entirely  freed 
from  fat.  All  the  precautions  that  were  taken  in  the  work  on  butter 
were  taken  here  also.     The  method  is  described  on  page  18. 

The  first  analyses  were  made  in  September  and  October,  1910. 
For  the  analyses  made  in  December,  1910,  and  in  June,  1911,  samples 
were  shipped  from  cold  storage  in  Chicage  by  express  to  Washington. 
Upon  their  arrival  the  material  was  at  once  transferred  to  the  re- 
frigerator in  the  Dairy  Division  laboratories,  where  it  remained  till 
used  for  analysis.  The  total  time  during  which  the  samples  were 
out  of  cold  storage  was  as  short  as  possible. 

Results. — From  the  results  obtained  on  samples  13,  15,  and  17, 
summarized  in  Table  3,  it  is  evident  that  in  buttermilk  obtained 
from  raw-cream  butter  the  activity  of  galactase  is  practically  en- 
tirely inhibited  by  the  presence  of  18  per  cent  of  sodium  chlorid  and 
by  the  low  temperature,  0°  F.  (  —  18°  C.)  of  the  cold  storage.  When 
some  of  this  same  material  is  allowed  to  remain  for  a  long  time  at 
room  temperature,  the  galactase  apparently  becomes  much  more 
active,  because  the  casein  is  seen  to  clot  and  the  mixture  assumes  the 
appearance  of  one  in  which  digestion  is  going  on. 

Samples  14,  16,  and  18  were  to  serve  as  controls  on  those  of  the 
other  three.  Although  galactase  in  cream  is  not  entirely  destroyed 
by  ordinary  pasteurization,  it  is  partly  inactivated.1  It  was  expected 
that  the  figures  for  water-soluble  nitrogen  in  samples  14,  16,  and  18 
would  change  little  during  storage,  thereby  affording  a  check  on  the 
correctness  of  the  work.  The  figures  for  water-soluble  nitrogen  in 
samples  13,  15,  and  17,  had  they  increased  during  storage,  could 
then  have  been  considered  as  obtained  by  a  method  that  showed  no 
change  where  none  is  to  be  expected. 

1  Rogers,  L.  A.,  Berg,  W.  N.,  and  Davis,  Brooke  J.    Loc.  cit.,  p.  318. 


PROTEOLYSIS  IN   MILK. 


29 


Table  3. — Effect  of  sodium  chlorid  and  cold  storage  (0°  F. 

galactose  in  buttermilk. 


■18°  C.)  upon  the  activity  of 


Buttermilk  obtained  from  churnings  of  unpas- 
teurized sweet-cream  buttei . 

Buttermilk  obtained  from  churnings  of  pas- 
teurized sweet-cream  butter. 

Butter- 
milk, 
lot  No. 
16.612, 
sample 
No. 

Butter, 
lot  No. 
10.311, 
sample 
No. 

N/5  water-soluble 

nitrogen  in  100  c.  c. 

buttermilk. 

10  per  cent 
acetic  acid 
used  as  pre- 
cipitant for 

Butter- 
milk, 

lot  No. 
10.622, 

sample 
No. 

Butter, 
lot  No. 
10.321, 
sample 
No. 

N/5  water-soluble 

nitrogen  in  100  c.  c. 

buttermilk. 

10  per  cent 
acetic  acid 
used  as  pre- 
cipitant for 

200  c.  c. 
buttermilk. 

Age. 

N/5N. 

200  c.c. 

buttermilk. 

Age. 

N/5  N. 

13 
15 
17 

42 
52 
65 

Days. 
25 

116 

301 

10 
124 
297 

10 
101 
286 

C.c. 
39.2 

36.8 
40.7 
41.0 
37.2 
37.4 
42.1 
40.9 
43.4 
42.6 
39.5 
40.0 
42.6 
45.3 
45.1 
46.3 
41.1 
38.7 

C.c. 
20 

18 
20 
18 
20 
18 
16 
20 
16 
20 
16 
20 
14 
12 
14 
12 
14 
12 

14 
16 

18 

40 
50 

62 

Days. 
25 

116 
301 

10 
124 
297 

10 
101 
286 

C.c. 
36.1 
38.2 
39.1 
36.3 
38.3 
31.1 
30.3 
31.9 
30.3 
30.1 
28.6 
35.0 
36.3 
35.2 
36.3 
33.6 
34.8 

C.c. 
18 
12 
12 
18 
12 
20 
24 
20 
24 
20 
24 
22 
18 
22 
18 
22 
18 

179  cubic  centimeters  N/5  nitrogen=average  total  nitrogen  in  100  cubic  centimeters  buttermilk. 

The  results  on  these  samples  of  buttermilk  confirm  the  results 
obtained  on  the  corresponding  samples  of  butter.  It  is  practically 
certain,  for  example,  that  butter  sample  No.  42  and  the  buttermilk 
obtained  from  it  both  contained  galactase,  though  in  different 
amounts.  Under  the  conditions  of  the  experiments  proteolytic  action 
was  uniformly  inhibited,  both  in  the  butter  and  in  the  buttermilk. 

As  the  following  calculations  show,  the  method  used  for  the  detec- 
tion of  proteolytic  action  in  buttermilk  (skim  milk)  is  quite  delicate 
and  should  lead  to  the  detection  of  proteolytic  action  were  it  appre- 
ciable. In  the  determination  of  water-soluble  nitrogen  in  buttermilk 
200  cubic  centimeters  were  taken,  which  contained  very  close  to  360 
cubic  centimeters  N/5  total  nitrogen.  Let  it  be  assumed  that  during 
the  cold  storage  period  5  per  cent  of  the  casein  became  water  soluble. 
Allowing  an  average  of  80  cubic  centimeters  N/5  nitrogen  in  water- 
soluble  form  originally  present  in  the  200  cubic  centimeters  of  butter- 
milk, there  would  be  formed  by  the  proteolysis  0.05X280  =  14  cubic 
centimeters  N/5  water-soluble  nitrogen.  In  the  actual  determination 
200  cubic  centimeters  of  buttermilk  were  diluted  to  500  cubic  centi 
meters  precipitated  with  acetic  acid,  and  two  200  cubic  centimeter 
portions  of  the  filtrate  were  used  for  nitrogen  determinations.  There- 
fore if  5  per  cent  of  the  casein  had  become  hydrolized  the  titrations 
after  cold  storage  would  have  been  5.6  cubic  centimeters  higher  than 
the  corresponding  titrations  before  storage.  It  is  probable  that  this 
increase  would  have  been  detected. 


30 


CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 


THE  INHIBITING  EFFECT  OF  SODIUM  CHLORID  AND  COLD  STORAGE 
UPON  THE  ACTIVITIES  OF  PROTEOLYTIC  ENZYMS  IN  STERILIZED 
SKIM  MILK. 

Description  of  samples. — Several  5-liter  flasks  full  of  skim  milk  were 
kept  in  a  steam  sterilizer  for  about  two  hours  at  a  temperature  vary- 
ing between  94°  and  99°  C. 

Three  3-liter  portions  were  measured  out  roughly  and  rapidly  while 
the  skim  milk  was  hot,  into  bottles,  samples  No.  20,  22,  and  24.  A 
weighed  quantity  of  the  enzym  preparation  was  then  added.  The 
amounts  are  given  in  Table  4,  page  30. 

After  cooling,  540  grams  of  sodium  chlorid  was  added  to  each 
sample.  The  bacterial  enzym  was  prepared  from  cultures  of  an  acid- 
forming  bacterium  that  secreted  a  proteolytic  enzym.  The  usual 
method  of  precipitating  with  alcohol,  etc.,  was  used.  The  dry  en- 
zym preparation  was  tested  before  it  was  used  in  the  experiments 
and  found  to  be  proteolytically  active.  The  other  enzym  prepara- 
tions were  the  ordinary  commercial  ones. 

Three-liter  portions  of  the  skim  milk  were  quickly  cooled  to  35°  C. 
To  each  was  added  540  grams  of  sodium  chlorid,  to  which  there  had 
been  previously  added  the  amount  of  enzym  indicated  in  the  table. 
Obviously,  samples  20,  22,  and  24  were  controls  on  Nos.  19,  21,  and  23. 

These  samples  were  canned  as  before.  Most  of  these  were  shipped 
to  cold  storage  in  Chicago,  where  they  were  maintained  at  a  tempera- 
ture of  20°  F.  (—  7°  C).  Determinations  of  water-soluble  nitrogen 
were  made  by  the  method  already  described  in  a  previous  publica- 
tion '  and  in  this  paper  page  18. 

Table  4. — Effect  oj  sodium  chlorid  and  cold  storage  upon  the  activities  of  proteolytic 
enzymes  in  sterilized  skim  milk  stored  at  20°  F.  (—7°  C). 


Skim 
milk, 
lot  No. 
10.612, 
sample 
No. 

Composition  of 
mixtures. 

N/5  water- 
soluble  ni- 
trogen s  in 
100  c.c.  skim 
milk  after 
75  days' 
storage. 

10  per  cent 
acetic  acid 
used  as  pre- 
cipitant for 
200c.c.skim 
milk. 

Skim 
milk, 
lot  No. 
10.622, 
sample 
No. 

Composition  of 

control 

mixtures. 

N/5  water- 
soluble  ni- 
trogen 2  in 
100  c.c.  skim 
milk  after 
77  days' 
storage. 

10  per  cent 
acetic  acid 
used  as  pre- 
cipitant for 
200  c.c.  skim 
milk. 

19 

Skim  milk,  3  liters 
Dairy    salt,    540 

grams. 
Bacterial 

C.c. 
44.9 
48.1 

C.  c. 
20 
10 

20 
22 
24 

Skim  milk,  3  liters 
Dairy   salt,    540 
grams. 

C.c. 

28.1 
33.1 

C.c. 

20 
10 

Enzym  (dry),  15 

grams. 
Skim  milk,  3  liters 
Dairy    salt,    540 

grams. 
Pancreatin,  3 

grams,  dry  (U. 

S.  P.). 
Skim  milk,  3  liters 
Dairy    salt,    540 

grams. 
Pepsin(U.S.P.), 

dry,  3  grams. 

Enzym  (boiled), 

15  grams. 
Skim  milk,  3  liters 
Dairy    salt,    540 

grams. 

21 

118.2 
121.0 

20 
10 

22.8                     20 

28.5  |                   10 

23 

54.1 
65.5 

20 

10 

P.),  boiled,  3 

grams. 
Slam  milk,  3  liters 
Dairy    salt,    540 

grams. 
Pepsin  (U.S.  P.), 

boiled,  3  grams. 

19.8 
24.2 

20 
10 

1  Rogers,  L.  A.,  Berg,  W.  N.,  and  Davis,  Brooke  J.    Loc.  cit.,  p.  315. 
*  198  c.  c.  N/5  nitrogen = average  total  nitrogen  in  100  c.  c.  skim  milk. 


PROTEOLYSIS  IN   MILK.  81 

Results. — In  sample  No.  19  there  was  present  a  large  quantity  of 
a  proteolytic  enzym  of  bacterial  origin  that  was  known  to  be  active 
on  gelatin.  It  is  highly  probable  that  much  more  enzym  was  present 
here  than  there  is  ever  present  in  butter,  and  the  figures  indicate 
plainly  that  the  salt  strongly  inhibited  its  action  during  the  period 
under  observation.  It  is,  of  course,  possible  that,  given  a  period  of 
time  greatly  exceeding  ordinary  storage  periods,  further  proteolysis 
in  this  sample  might  have  been  observed. 

In  samples  21  and  23  digestion  took  place  to  a  large  extent.  In 
No.  21  approximately  two-thirds  of  the  total  protein  had  become 
water  soluble. 

On  samples  20,  22,  and  24  practically  identical  results  were  ob- 
tained before  and  after  storage,  as  would  be  expected.  This  indi- 
cated that  in  the  controls  no  proteolytic  changes  were  detected. 

CONCLUSIONS. 

It  is  evident  from  these  results  that  in  the  presence  of  very  large 
amounts  of  strongly  active  proteolytic  enzyms,  proteins  will  be 
hydrolized  even  in  cold  storage  (or  in  transit)  and  in  strong  salt  solu- 
tions. But  there  is  no  reason  to  suppose  that  at  any  time  such 
amounts  of  enzyms  are  ever  found  in  butter. 

It  is  very  probable  that  samples  21  and  23  contained  several  thou- 
sand times  as  much  proteolytic  enzym  as  is  present  in  ordinary 
butter  of  any  kind. 

However,  it  must  be  borne  in  mind  that  the  claim  is  not  made 
that  sodium  chlorid  does  not  exert  an  inhibiting  influence  on  pro- 
teolytic action.  Whether  it  does  or  does  not  depends  upon  the 
conditions  of  the  experiment.  It  is  comparatively  easy  to  show 
that  the  action  of  pepsin-acid  can  be  inhibited  very  strongly  by 
large  amounts  of  sodium  chlorid.  In  the  spring  of  1909  some  experi- 
ments were  made  in  which  the  speed  of  digestion  of  casein  in  several 
pepsin-acid  solutions  was  compared  with  that  in  the  same  solutions 
to  which  20  grams  of  sodium  chlorid  to  100  cubic  centimeters  of  acid 
solution  had  been  added.  The  presence  of  the  salt  almost  completely 
inhibited  the  action  of  the  pepsin-acid  during  the  time  of  the  experi- 
ment, 40  minutes'  digestion,  but  it  is,  of  course,  possible  that  proteo- 
lytic action  would  have  taken  place  had  the  digestion  period  been 
several  months.  The  method  of  comparing  speeds  of  digestion  was 
that  described  by  Gies.1  In  general,  the  statement  that  sodium 
chlorid  does  inhibit  proteolysis  is  true,  therefore,  at  low  tempera- 
tures, when  the  amount  of  enzym  is  small  and  the  digestion  period 
(storage  period)  is  long  or  when  the  amount  of  enzym  is  large  and 
the  digestion  period  is  short,  as  in  ordinary  digestion  experiments. 

1  Berg,  William  N.,  and  Gies,  William  J.  Studies  of  the  effects  of  ions  on  catalysis,  with  particular 
reference  to  peptolysis  and  tryptolysis.  Journal  of  Biological  Chemistry,  vol.  2,  no.  6,  pp.  489-546.  New 
York,  March,  1907. 


32  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

The  statement  that  sodium  chlorid  does  not  inhibit  proteolytic 
action  is  true  at  comparatively  high  temperatures  when  the  amount 
of  enzym  is  very  large  and  the  digestion  period  very  long.  The 
apparently  contradictory  statements  are  the  results  of  testing  the 
action  of  the  sodium  chlorid  over  a  wide  range  of  enzym  concentra- 
tion and  over  widely  varying  digestion  periods. 

Several  investigators  have  studied  the  action  of  sodium  chlorid  on 
the  tryptic  digestion  of  casein.  Their  conflicting  statements  are,  of 
course,  easily  accounted  for  by  the  fact  that  the  experiments  were 
made  under  conditions  that  were  not  uniform  with  regard  to  the 
concentration  of  sodium  chlorid,  the  relative  proportions  of  alkali, 
trypsin,  and  casein,  etc.     Their  work  is  summarized  by  Robertson.2 

THE    INDIRECT    ACTION    OF   BACTERIA. 

The  improbability  that  the  proteolytic  enzyms  are  responsible  for 
the  difference  in  keeping  quality  between  unpasteurized  and  pasteur- 
ized sweet-cream  butter  is  shown  by  the  work  given  in  detail  in  the 
preceding  pages.  The  bacteria  are  another  possible  factor  removed 
by  pasteurization.  While  it  is  certain  that  they  do  not  grow  at  the 
low  temperature  of  commercial  storage,  it  is  possible  that  the  pres- 
ence of  a  large  number  of  living  cells,  or  various  active  enzyms 
which  may  possibly  be  liberated  by  the  death  of  the  bacteria,  may 
have  an  influence  on  the  flavor  of  the  butter.  If  the  bacteria 
destroyed  by  the  pasteurization  could  be  replaced  in  the  cream  their 
influence  on  the  flavor  should  be  shown  in  a  comparison  of  the  butter 
made  from  this  reinoculated  cream  and  that  made  from  a  part  of  the 
same  pasteurized  cream,  but  without  the  addition  of  bacteria. 
Before  this  could  be  done  intelligently  it  was  necessary  to  obtain  a 
general  knowledge  of  the  bacteriological  content  of  the  raw  cream. 
The  normal  bacteria  of  the  cream  from  one  skimming  station  was  deter- 
mined by  sampling  every  day  and  plating  on  lactose  agar.  After  7 
days'  incubation  at  about  30°  C.  the  plates  were  counted  and  all  of 
the  colonies  on  a  representative  plate  were  transferred  to  litmus  milk 
tubes.  These  were  incubated  and  examined  after  2,  5,  and  14  days. 
This  enabled  a  separation  into  high  acid  forms  which  curdled  the  milk 
in  less  than  2  days;  low  acid  forms  forming  acid  but  curdling  the 
milk  slowly  or  not  at  all ;  alkali  formers,  peptonizers,  and  those  that 
produce  no  visible  change  in  the  milk.  These  groups  were  calculated 
as  percentages  of  the  total  bacteria,  and  the  results  are  given  in 
Table  5. 

*  Robertson,  T.  Brailsford.  On  some  chemical  properties  of  casein  and  their  possible  relation  to  the 
chemical  behavior  of  other  protein  bodies,  with  especial  reference  to  hydrolysis  of  casein  by  trypsin. 
Journal  of  Biological  Chemistry,  vol.  2,  no.  4,  pp.  317-383.    New  York,  January,  1907.    See  p.  355. 


THE  INDIRECT   ACTION   OF  BACTERIA.  33 

Table  5. — Numbers  of  bacteria  with  distribution  in  different  groups  in  cream. 


No. 

Bacteria  per 
cubic  centi- 
meter. 

High.acid. 

Low  acid. 

Alkali. 

Pepton- 
izers. 

No  change. 

1 
2 
3 
4 
5 
6 
7 
8 
9 

10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
25 
26 

116,000,000 
61,000,000 
23,500,000 
40,000,000 
99,000,000 
34,000,000 
11,800,000 
33,500,000 
51,000,000 
40,000,000 
8,000,000 
21,800,000 
37,000,000 

101,000,000 

147,000,000 
54,500,000 
69,500,000 
30,000,000 
89,000,000 
98,000,000 

110,000,000 
45,000,000 
52,000,000 
87,500,000 
39,500,000 
5,700,000 

Per  cent. 
25.8 
41.6 
21.2 
48.8 
14.1 
36.1 
16.3 
49.0 
27.1 
9.3 
27.9 
20.4 
27.8 
68.7 
60.2 
36.0 
29.5 
51.7 
42.9 
15.3 
15.0 
12.6 
30.0 
6.8 
7.5 
33.*3 

Per  cent. 
11.3  v 
16.6 
7.7 
2.4 
9.9 
8.4 
12.1 
23.0 
14.6 
2.3 
8.9 
3.3 

Per  cent. 
1.0 
6.7 
1.0 

Per  cent. 

1.0 
10.0 

7.3 
12.2 
25.3 

Per  cent. 
60.8 
25.0 
62.1 
36.6 
50.5 
55.6 
55.9 
25.7 
54.2 
80.0 
55.7 
70.3 
61.1 
18.7 
24.7 
46.0 
47.5 
24.1 
46.2 
84.7 
78.0 
68.5 
46.0 
87.5 
80.0 
64.9 

1.1 

1.7 

12.9 
3.0 
4.2 
7.2 
1.3 
6.4 
8.3 
44 
1.8 
6.0 
3.2 
3.4 
1.1 

6.3 

2.8 
4.4 
5.3 
6.0 
8.2 
3.4 
2.2 

3.6 
8.0 
6.0 
11.5 
17.2 
7.7 

7.0 
18.9 
12.0 
5?7 
7.5 
1.8 

12.0 

5.0 

It  will  be  noticed  that  the  number  of  bacteria  in  the  cream  was 
high  and  that  there  was  great  variation  in  the  proportion  of  the  groups 
from  day  to  day. 

REINOCULATION    OF    CREAM. 

Typical  cultures  were  saved  from  each  group  and  used  to  inoculate 
one-half  of  a  lot  of  pasteurized  cream.  Sufficient  amounts  of  milk 
cultures  were  added  to  the  pasteurized  cream  to  bring  the  bacterial 
count  at  the  end  of  about  *24  hours  to  numbers  and  proportions 
approximately  those  of  the  cream  before  pasteurization.  This  was 
of  course  very  difficult  to  control,  but  the  results  given  in  Table  2 
show  that  this  was  attained  within  reasonable  limits.  Two  experi- 
ments of  this  kind  were  made,  each  consisting  of  three  lots  of  butter. 
One-third  of  a  lot  of  sweet  cream  was  cooled  and  churned;  two- 
thirds  was  pasteurized  by  holding  at  145°  F.  for  20  minutes;  one- 
half  of  the  pasteurized  cream  was  cooled  and  churned,  and  the 
remaining  portion  inoculated  with  the  milk  cultures  and  held  about 
24  hours  to  allow  bacteria  to  develop.  There  was  no  appreciable 
increase  in  acidity  in  this  period.  The  cream  was  then  cooled  and 
churned  as  before.  It  is  probable  that  the  lipase  was  destroyed  and 
the  galactase  weakened.  Tests  showed  that  catalase  was  present  in 
the  raw  cream,  absent  in  the  pasteurized  cream,  and  present  again 
in  the  inoculated  cream,  while  all  three  lots  gave  a  test  for  peroxidase. 
66711°— Bull.  162—13 5 


34 


CHANGE   IN    FLAVOR    OF    STORAGE   BUTTER. 


The  score  of  the  butter  given  in  Table  7  shows  that  while  there 
was  a  marked  difference  in  the  rate  of  deterioration  in  the  raw-cream 
butter  and  the  pasteurized-cream  butter,  the  reinoculation  of  the 
cream  with  bacteria  had  little  or  no  effect  on  the  keeping  quality  of 
the  butter.  In  one  case  the  inoculated-cream  butter  changed  more 
than  the  uninoculated,  while  in  the  other  lot  the  reverse  was  true. 

Table  6. — Bacteria  in  cream  vised /or  making  butter. 
No.  l. 


Bacteria  per 
Treatment  of  cream.                  cubic  centi- 
meter. 

High 
acid. 

Low 
acid. 

Alkali. 

Pepton- 
izes. 

No 
change. 

Raw 48,000,000 

Pasteurized 53.000 

Per  cent. 
13.1 

Per  cent. 
10.9 

Per  cent. 
0.0 

Per  cent. 
17.4 

Per  cent. 
58.6 

20,000,000 

18.7 

6.6 

0.0 

2.5 

72.2 

No.  2. 

47,000,000 

23,400 

217,000,000 

46.0 

6.3 

0.0 

6.3 

41.4 

26.8* 

.5 

1.0 

1.0 

70.7 

Table  7. — Scores  oj  butter — raw,  pasteurized,  and  reinoculated  cream. 

Treatment  of  cream. 


First  score. 


days. 


Raw 

Pasteurized 

Pasteurized  and  inocu 
lated. 

Raw 

Pasteurized 

Pasteurized  and  inocu 
a  ted. 


Total 
score. 


91 


Comments. 


Very    unclean,  some- 
what rancid. 
Slightly  woody. ....... 

Woody 

Unclean,  rancid 

Slightly  woody 

Woody 


Second  score. 


Age, 
days. 


36 


Total 
score. 


S2 


Comments. 


Rank,  woody, rancid. 

Very  woody. 
Somewhat  woody. 

Rancid,  woody. 
Slightly    unclean, 

woody. 
Woody. 


It  is  perhaps  significant  that  raw,  sweet-cream  butter  always 
becomes  rancid,  a  flavor  usually  associated  with  the  liberation  of 
fatty  acid.  It  is  not  improbable  that  the  lipase  of  the  milk  is  able  to 
act  under  these  conditions,  but  that  the  ripening  of  raw  cream,  which 
usually  prevents  the  development  of  this  flavor,  inhibits  the  action 
of  this  enzym. 

THE  POSSIBLE  OXIDATION  OF  BUTTER  BY  INCLOSED  AIR. 

The  possible  oxidation  of  butter  by  finely  divided  air  globules 
inclosed  in  it,  as  previously  pointed  out,  made  it  desirable  to  find 
out  how  much  air  was  present  in  butter  exclusive  of  large  pockets, 
and  second,  whether  the  air  present  in  butter  underwent  a  change 
during  storage  through  the  transfer  of  oxygen  from  the  air  to  some 


THE   POSSIBLE   OXIDATION   OF   BUTTER   BY  INCLOSED  AIR. 


35 


oxidizable  substance  in  the  butter.     The  work  was  done  in  the  field 
laboratory  of  the  Dairy  Division,  Troy,  Pa. 

Method  of  gas  analysis. — The  following  diagram  shows  the  appa- 
ratus used  for  obtaining  the  gas  from  a  can  of  butter.  The  deter- 
mination of  the  amount  of  air  (or  gas)  in  a  can  of  butter  was  made 
as  follows :  The  can  of  butter  was  removed  from  the  refrigerator  late 
in  the  afternoon  and  allowed  to  remain  at  room  temperature  over 
night.  The  next  morning  the  can  was  placed  in  water  at  45°  C.  for 
about  an  hour  until  the  contents  of  the  can  were  melted.  The  flasks, 
previously  connected,  and  the  Toepler  pump,1  were  then  evacuated 
by  the  Geryk  pump  through  the  tube  d.  Tube  /  could  be  evacuated 
by  opening  the  stopcock  toward  the  Toepler  pump.  The  Geryk 
pump  was  worked  until  the  McLeod  gauge  on  the  Toepler  pump 
read  about  0.3  millimeter.     The  flask  i  and  tube  j  were  then  evac- 


7b  Geryk  pump 


7b  7oep/er-  pump 


uated.  The  can  punch  was  then  brought  under  tube  g  and  the 
can  of  butter  placed  inside  of  n.  The  brass  plate  q,  carrying  the 
upper  needle,  was  placed  over  the  can.  The  plate  is  held  over  the 
can  by  two  brass  guide  rods,  threaded  at  their  upper  ends  and 
screwed  into  r,  the  brass  base,  q  was  then  screwed  down  a  short 
distance,  so  that  the  upper  end  of  the  upper  needle  I  could  be  brought 
under  the  lower  end  of  tube  g.  I  and  g  were  connected  by  the 
usual  rubber  tube  and  rubber  nipple  mercury  seal.  Water  at  60°  C. 
was  poured  into  the  container  until  the  can  was  completely  sub- 
merged. The  water  was  then  colored  by  adding  a  few  cubic  centi- 
meters of  alcoholic  solution  of  methylene  blue.  Previous  to  putting 
the  can  of  butter  into  n,  the  funnel  p  was  filled  with  a  three-fourths 
saturated  sodium  chlorid  solution  at  50°  C.  and  air  bubbles  removed 
from  o,  partly  by  squeezing  o  and  forcing  the  air  bubbles  up  to  p, 

1  We  used  an  improved  form  of  Toepler  pump  designed  by  Dr.  Clark,  of  this  laboratory. 


36  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

and  then  by  opening  the  screw  clamp  at  t  and  allowing  the  solution 
to  flow  through  V  into  n.  The  two  needles  were  forced  into  the  can 
by  screwing  down  q.  In  order  not  to  open  the  mercuiy  seal  at  the 
upper  end  of  I,  the  entire  punch  was  raised  as  fast  as  I  was  lowered, 
by  means  of  several  thin  wooden  strips  and  a  wedge  on  both  sides. 
When  it  was  apparent  that  the  rubber  stoppers  &  were  pressing  very 
tightly  against  the  can,  and  very  soon  the  needles  would  punch  the 
can,  I  and  g  were  evacuated  by  opening  the  stopcock  toward  i. 
The  entire  system  was  now  empty,  from  I  to  g,f,  c,  b,  and  the  Toepler 
pump.  The  Geryk  pump  was  then  cut  off  altogether  from  the  rest 
of  the  system,  q  was  still  further  forced  down  until  the  lower  and 
then  the  upper  needles  pierced  the  can.  When  the  upper  cover  was 
pierced,  butter  fat  would  fill  tube  g  to  stopcock  h'.  This  was  then 
opened  and  butter  fat  was  slowly  passed  through  /  into  c,  where  it 
remained.  The  inclosed  gases  passed  on  through  b  to  the  Toepler 
pump.  The  screw  clamp  t  was  removed  soon  after  the  butter  began 
to  flow  out  of  the  can,  permitting  salt  solution  to  enter  the  can  as 
fast  as  butter  left  it.  During  the  passage  of  butter  fat,  and  later  of 
butter  curd  solution,  the  entire  can  was  covered  with  water.  In  only 
one  experiment  did  any  colored  solution  find  its  way  into  the  tube  g. 
Stopcock  A'  shut  off  the  connection  between  g  and/ when  salt  solution 
began  to  come  through  g.  This  usually  happened  when  most  of  the 
butter  fat  and  part  of  the  curd  solution  had  passed  into  c.  Knowing 
the  weight  of  the  can  of  butter,  the  empty  can,  the  weight  of  c 
empty  and  when  filled  with  butter  from  the  can,  the  amount  of 
butter  passed  into  c  could  be  ascertained  approximately.  In  general 
between  seven  and  eight  tenths  of  the  contents  of  the  can  were  passed 
into  c  during  the  experiment.  The  gas  in  the  Toepler  pump  and 
flasks  was  then  pumped  out  in  the  usual  way  and  measured.  Part  of 
it,  50  to  60  cubic  centimeters,  was  used  for  the  determination  of  cai- 
bon  dioxid  and  of  oxygen.  The  usual  apparatus  was  used.  The 
determinations  were  made  according  to  the  methods  described  in 
Hempel's  Methods  of  Gas  Analysis,  1906,  pages  149  and  201. 

One  blank  on  air  was  made  before  the  results  were  obtained,  one 
after,  and  two  between  results,  so  that  the  reagents  were  certainly  in 
good  condition. 

In  the  third  determination  on  2A  a  few  drops  of  colored  water  were 
seen  to  pass  up  the  tube  g.  No  more  butter  (butter  fat)  was  allowed 
to  pass  into  c  and  the  estimation  of  the  quantity  and  composition  of 
the  gas  was  made  as  usual.  It  is  probable  that  the  apparently  high 
carbon  dioxid  was  due  to  greater  volatilization  of  acid-reacting 
material.  In  this  experiment  flask  c  contained  525  grams  of  butter 
instead  of  the  usual  700-800  grams. 

Results. — The  butter  used  had  been  churned  from  pasteurized 
ripened  cream,  packed  carefully  in  2-pound  tin  cans,  and  sealed  by  a 


THE   POSSIBLE    OXIDATION    OF    BUTTER    BY   INCLOSED   AIR. 


37 


sealing  machine.  Determinations  were  made  before  and  after  stor- 
age. Part  of  the  butter  was  worked  normally  in  the  churn  (samples 
marked  "A"),  part  was  overworked  (samples  marked  "B").  Sam- 
ples 1A  and  IB  were  obtained  from  the  same  lot  of  butter  and  samples 
2A  and  2B  were  from  another  lot. 

Table  8. — Quantity  and  composition  oj  gas  in  pasteurized  ripened-cream  butter. 

BEFORE  STORAGE. 


Volume  of  gas 
obtained  from 
1  can  (capacity 
approximately 
1  liter)  of  but- 
ter reduced  to 
0°C.  and  760 
mm.  of  mer- 
cury. 

Composition  of  gas. 

Lot  No.  19.4. 

Carbon 
dioxid . 

Oxygen. 

Sample  No. — 

.  c. 

99.6 

•       101.4 

107.7 

98.8 
141.5 
104.6 
101.4 

Per  cent. 

Per  cent. 

1A      

31.3 
29.7 
36.8 
37.8 
37.8 
.8 

19.1 

IB 

20.3 

2A • 

19.1 

2B 

19.5 

2B 

18.6 

21.87 

AFTER  STORAGE. 


Sample  No.— 

1A 

94.1 
76.9 
139.0 
112.0 
71.0 
117.5 

34.2 
32.5 
31.5 
39.3 
52.8 
33.6 
.38 
.39 
.39 
.66 

13.3 

IB 

11.0 

2A 

13.1 

2A...              

12.1 

2A 

10.0 

2B 

13.3 

22.1 

21.5 

20.77 

21.82 

The  results  (see  table  8)  before  storage  were  obtained  in  November 
and  December,  1911.  All  of  the  results  for  carbon  dioxid  were 
obtained  on  the  same  pipetteful  of  potassium  hydroxid  solution  and 
all  of  those  for  oxygen  on  the  same  pipetteful  of  sodium  pyrogallate. 
The  blank  was  made  after  all  the  other  results  had  been  obtained. 
Two  separate  determinations  on  two  cans  of  2B  showed  that  the  same 
result  could  be  obtained  on  two  cans  of  the  same  lot  of  butter. 

On  the  assumption  that  the  gas  in  butter  is  a  mixture  of  carbon 
dioxid  and  air,  the  oxygen  content  found  is  apparently  high. 

If  the  percentage  of  carbon  dioxid  be  subtracted  from  100,  leaving, 
for  example,  70  per  cent  of  the  gas  supposedly  air,  the  percentage  of 
oxygen  (20)  forms  too  large  a  part  of  the  total  volume  of  gas  from 
which  carbon  dioxid  has  been  removed. 

The  butter  was  stored  at  0°  F.  until  the  following  March,  1912, 
during  which  month  the  results  after  storage  were  obtained. 

The  results  are  difficult  to  interpret,  partly  because  very  few  of 
them  have  as  yet  been  obtained  and  partly  because  certain  check 


38  CHANGE   IN   FLAVOR   OP   STORAGE   BUTTER. 

experiments  not  yet  made  will  be  necessary.  There  seems  to  be  n< 
great  variation  in  carbon  dioxid  content,  but  a  decided  lowering  of 
the  oxygen  content  as  if  part  of  this  gas  had  been  removed.  It  is 
possible  that  further  work  will  show  that  the  oxygen  in  these  experi- 
ments was  actually  transferred  to  some  oxidizable  substance  in  the 
butter.  It  would  perhaps  be  better  to  defer  conclusions  until  further 
work  shows  that  the  oxygen,  if  it  was  indeed  removed,  went  to  some 
butter  constituent  and  was  not  combined  with  metal  or  diffused  out 
through  the  seals.  It  seems  highly  desirable  to  ascertain  definitely 
whether  cans  sealed  by  a  machine  without  the  use  of  solder  are  gas 
tight,  measuring  gas  tightness  for  a  period  of  several  months. 

The  cans  used  were  heavily  tinned  and  well  lacquered  on  the  inside. 
The  rims  of  the  covers  were  provided  with  a  layer  of  rubber  cement, 
which  was  forced  into  the  seal  by  the  sealing  machine.  Cans  sealed 
in  this  way  are  air  tight  when  tested  under  15  pounds'  pressure  for  a 
short  time. 

The  figures  for  the  composition  of  the  gases  in  butter  obtained  after 
storage  show  that  the  gas  is  apparently  a  mixture  of  carbon  dioxid 
and  air.  At  present  any  conclusion  regarding  the  nature  of  the  gas 
is  premature.  It  seems  almost  certain  that  the  gas  in  these  samples 
was  not  a  mixture  of  carbon  dioxid  and  air  only,  but  that  some  vola- 
tile substance  was  mixed  with  it.  The  gas  obtained  from  butter  had 
an  intensely  "buttery"  odor.  Perhaps  a  more  detailed  analysis  of 
the  gas  will  make  the  results  more  intelligible.  Under  the  circum- 
stances it  is  not  safe  to  assume  that  the  figures  for  carbon  dioxid 
represent  carbon  dioxid  alone,  for  any  substance  having  an  acid 
character  and  volatile  under  the  conditions  of  the  experiment  would 
probably  be  included  with  the  carbon  dioxid  in  the  potassium 
hydroxid  absorption.  These  results  show  that  butter  contains 
about  10  per  cent  by  volume  of  gases. 

Overworked  butter  did  not  contain  any  more  air  than  that  which 
had  been  normally  worked.  For  obvious  reasons  these  results  are 
not  comparable  with  those  obtained  by  overworking  small  amounts 
of  butter  with  a  spatula.1  It  may,  however,  be  significant  that  the 
decrease  in  oxygen  as  shown  in  Table  8  was  about  50  per  cent  greater 
in  the  overworked  sample,  IB,  than  in  any  other. 

THE  EFFECT  OF  METALS  ON  BUTTER. 
EARLIER   INVESTIGATIONS. 

The  influence  of  metals  on  the  changes  in  butter  has  received  some 
attention,  although  most  of  the  experiments  along  this  line  have  not 
included   storage  butters.     In    1902    Henzold 2  found   butter   with 

l  Rogers,  L.  A.    Fishy  flavor  in  butter.    United  States  Department  of  Agriculture,  Bureau  of  Animal 
Industry,  Circular  146.    Washington,  1909. 
*  Henzold,  Ottomar.    Bittere  Butter.    Milch-Zeitung,  vol.  31,  no.  52,  pp.  822-823,  Leipzig,  pec.  27, 1902. 


THE    EFFECT    OF    METALS    ON   BUTTER.  39 

a  bitter  astringent  taste,  which  he  concluded  was  caused  by  iron  in  the 
salt.  He  made  butter  from  pasteurized  cream,  to  which  salt  con- 
taining 0.05  to  0.1  per  cent  iron  oxid  was  added.  The  butter 
made,  using  this  salt,  had  a  decidedly  bitter  taste.  By  elimination 
and  control  of  conditions,  the  iron  was  found  to  be  the  factor  influ- 
encing this  taste. 

L.  Marcas  1  showed  the  effect  of  holding  milk  and  cream  in  rusted 
cans  for  from  2  to  46  hours  and  making  butter  from  the  cream 
treated  in  this  manner.  He  determined  the  amount  of  iron  in  the 
milk,  skim  milk,  cream,  butter,  and  buttermilk,  holding  part  in  a 
clean  can  and  the  other  in  a  rusted  can  for  comparison.  He  found  in 
all  cases  a  bitter,  astringent  taste  and  bad  odor  in  butter  made  from 
milk  held  in  rusted  cans,  while  the  butter  made  from  the  milk  held  in 
clean  cans  was  of  good  quality.  He  concluded  that  the  milk  coming 
in  contact  with  the  iron  rust  forms  iron  lactate  from  the  iron  oxid  and 
that  it  is  the  lactate  which  causes  the  bitter  taste.  This  solvent 
action  he  found  to  be  especially  active  in  cieam,  owing  to  its  acidity. 
He  found  that  cream  with  a  normal  iron  content  of  0.005  parts  per 
1,000  would  increase  to  0.240  by  22  hours'  contact  with  a  rusted  can 
and  to  0.270  by  46  hours'  contact.  Butter  made  from  the  cream 
containing  0.240  parts  per  1,000  contained  0.080  parts  of  iron  per 
1,000,  while  butter  made  from  cream  containing  0.270  parts  per  1,000, 
contained  0.134  parts  of  iron  per  1,000. 

Hoft 2  in  1909  added  iron  salts  (ferrous  ammonium  sulphate  and 
iron  lactate)  to  cream,  allowed  the  cream  to  stand  up  to  22  hours, 
made  butter,  scored  it  for  physical  changes,  and  made  qualitative 
tests  for  iron  in  the  butter.  He  added  iron  in  quantities  ranging  from 
2  parts  per  1,000,000  to  33  parts  per  1,000,000.  He  published  results 
of  only  eight  tests,  which  showed  in  most  cases  an  oily  metallic  taste 
in  the  butter  and  in  which  the  presence  of  iron  in  the  curd  solution 
was  determined  with  potassium  sulphocyanid.  He,  however,  cau- 
tioned against  definite  conclusions,  as  he  had  not  decided  that  the 
change  was  due  to  iron  unconditionally.  He  found  that  small 
amounts  of  iron  acting  for  a  long  time  caused  more  effect  than  large 
quantities  of  iron  for  a  short  time. 

In  1911  the  Molkerei-Zeitung  3  published  some  work  on  the  effect 
on  butter  of  washing  it  with  water  containing  9  to  15  milligrams  iron 
per  liter.  This  caused  the  butter  to  have  a  metallic,  oily,  tallowy 
taste.  The  butter  when  washed  with  water  from  which  the  iron  had 
been  removed  by  oxidation  and  filtration  did  not  show  the  faults 

1  Marcas,  L.,  and  Huyge,  C.  Influence  de  la  rouille  sur  la  quality  du  beurre.  L'Industrie  Laitiere, 
vol.  30,  no.  16,  pp.  187-188.    Paris,  Apr.  16, 1905. 

2  H6ft,  Dr.  Kann  man  aus  dem  chemischen  Nachweis  von  Eisen  in  der  Butter  auf  eine  Qualitatsver- 
minderung  der  Butter  dureh  das  Eisen  schliessen?  Milchwirtschaftliches  Centralblatt,  vol.  5,  no.  6,  pp 
250-252.    Leipzig,  June,  1909. 

»  Enteisenungsanlage  fur  Molkereiwasser.  Molkerei-Zeitung,  vol.  25,  no.  58,  pp.  1095-1096.  Hildesheim, 
July  28,  1911. 


40  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

mentioned.  It  was  also  found  that  holding  cream  in  rusted  vessels 
caused  this  oily,  metallic  taste. 

Kooper,1  in  1911  did  some  work  to  show  that  washing  butter 
with  water  containing  pure  metallic  iron  would  not,  in  quantities  up 
to  36  milligrams  per  liter,  cause  any  noticeable  changes  in  the  quality 
of  the  butter,  but  that  the  changes  that  took  place  were  caused  by 
othei  substances  in  the  water  together  with  the  iron.  He  is  of  the 
opinion  that  water  containing  a  high  percentage  of  iron  is  very  likely 
to  contain  H2S  or  nitrous  acid,  which  would  be  more  likely  to  cause 
the  changes  and  defects  in  the  butter  than  the  iron  itself.  Kooper 
used  a  saccharated  iron  carbonate  in  his  work.  He  says,  however, 
that  a  change  of  the  iron  to  lactate  is  caused  by  contact  of  high  acid 
cream  with  iron,  and  this  will  produce  the  oily  and  metallic  flavors. 
According  to  Kooper  the  iron  taken  up  depends  on  the  length  of  time 
of  contact  and  the  acidity  of  the  cream.  By  adding  rusted  nails  or 
pulverized  iron  rust  to  the  cream  before  ripening  and  allowing  the 
cream  to  ripen  in  contact  with  the  iron  rust  he  found  the  cream  to  be 
changed  to  a  grayish  color  and  showing  defects  in  odor  and  taste. 
He  also  showed  that  the  washing  tended  to  take  some  of  the  iron  from 
the  butter. 

A  summary  of  the  preceding  work,  then,  shows  that  some  investi- 
gators think  the  iron  in  wash  water  (9  to  15  milligrams  per  liter)  is 
responsible  for  the  changes  in  butter,  while  others  do  not  think  the 
iron  alone  (up  to  36  milligrams  per  liter)  will  cause  the  changes.  All 
seem  to  agree  that  the  contact  of  acid  cream  will  take  up  iron  from 
rusted  containers,  change  it  to  a  lactate,  and  produce  butter  of  poor 
flavor. 

In  order  to  see  whether  butter  containing  iron  would  change  more 
in  storage  than  clean  butter,  it  became  necessary  to  make  butter 
which  contained  no  impurity  or  foreign  matter  other  than  iron  and 
also  to  make  a  control  butter  containing  no  foreign  material  and 
free  from  iron. 

Anyone  familiar  with  creamery  methods  will  readily  see  that  to 
make  butter  free  from  contact  with  iron  would  require  careful  super- 
vision of  the  cream  from  the  time  of  milking  to  the  making  of  the 
butter.  In  order  to  do  this,  the  cream  was  all  selected  from  farmers 
who  were  careful  in  the  handling  of  the  cream  and  whose  cans  were 
free  from  rust,  as  this  is  the  first  opportunity  for  contact  with  iron. 
The  cream  was,  in  most  cases,  pasteurized  in  a  Jensen  flash  pasteur- 
izer at  75°  C.  This  was  carefully  cleaned.  The  cream  was  cooled, 
weighed,  and  then  ripened  in  enamel-lined  tanks  free  from  iron. 
These  tanks  were  made  especially  for  this  work  to  eliminate  any 
chance  for  contact  with  iron  during  ripening,  at  which  time,  owing 

1  Kooper,  W.  D.    1st  der  Eisengehalt  des  Wassers  von  Einfluss  auf  die  Qualitat  der  Butter?    Milch- 
Zeitung,  vol.  40,  no.  29,  pp.  285-287.    Leipzig,  July  22,  1911. 


THE   EFFECT   OF    METALS   ON   BUTTER.  41 

to  the  acidity  of  the  cream,  the  cream  would  be  most  likely  to  attack 
and  dissolve  the  iron.  A  pure  culture  starter  was  used,  the  same 
precautions  in  regard  to  metals  being  taken  in  the  preparation  of  the 
starter.  After  pasteurizing,  the  cream  was  divided  into  two  portions, 
each  being  put  into  an  enamel-lined  tank  for  ripening.  The  cream 
was  cooled  by  running  brine  through  tinned  copper  coils  so  suspended 
in  the  vats  that  they  could  also  be  used  for  stirring  and  mixing. 
The  cream  in  one  vat  was  held  under  normal  conditions  and  free 
from  iron.  To  the  cream  in  the  second  vat  there  were  added  known 
amounts  of  iron.  Both  ferrous  sulfate  and  ferrous  lactate  were 
used  in  this  work.  This  iron  was  added  in  amounts  varying  from 
1  to  500  parts  of  iron  per  million  of  cream  (or  1  to  500  milligrams  of 
iron  in  a  kilo  of  cream).  The  two  vats  of  cream  were  then  ripened 
under  like  conditions  and  churned  at  the  same  time  in  the  Disbrow 
combined  churns  and  workers  (B2,  of  50  gallons  capacity).  These 
churns  were  shipped  to  Troy,  Pa.,  from  Albert  Lea,  Minn.,  in  the 
spring  of  1911  and  were  exposed  for  some  time.  Before  using  at 
Troy  they  were  taken  apart,  scraped,  sandpapered,  and  thoroughly 
cleaned,  though  in  spite  of  this  one  iron  plate  at  the  side  showed 
rust  which  was  exposed  to  the  cream.  The  butters  were  worked 
the  same  amount,  salted  the  same,  and  washed  with  the  same  amount 
of  water  and  the  same  number  of  revolutions  in  wash  water.  This 
was  considered  an  essential  point,  as  Kooper  found  that  butter  that 
was  washed  and  worked  held  less  iron  than  that  which  was  worked 
without  washing.  The  butters  were  then  carefully  packed  in  glass 
jars,  with  glass  tops,  to  avoid  any  contact  with  metals,  and  placed  in 
storage.  The  butters  made  at  Albert  Lea,  Minn.,  were  scored  about 
a  week  after  making  and  again  after  three  months'  storage  at  10°  F. 
This  butter  was  scored  by  J.  B.  Neumann,  J.  C.  Joslin,  P.  H.  Keiffer, 
and  the  Fox  River  Butter  Co.,  none  of  whom  were  familiar  with  the 
history  of  the  butter.  As  these  butters  were  shipped  to  several  cities, 
it  was  impossible  to  get  scores  on  the  day  the  butters  were  made, 
the  time  usually  approximating  one  week. 

The  butter  made  at  Troy,  Pa.,  was  scored  by  Mr.  Fryhofer  and 
Mr.  Smarzo,  of  New  York  City,  after  from  two  to  four  days,  again 
after  about  a  month,  and  again  after  about  three  months'  storage  at 
from  6°  to  10°  F.  By  comparison  of  the  scores  of  the  control  butters 
and  those  to  which  iron  had  been  added  at  the  different  intervals  the 
effect  of  the  iron  on  the  quality  of  the  butter  was  determined  by  the 
score  for  flavor,  aroma,  and  body. 

In  order  to  determine  the  relation  between  the  amount  of  iron 
present  in  the  butter  and  the  deterioration  of  the  butter,  it  became 
nepessary  to  know  the  amount  of  iron  present,  as  well  as  the  changes 
shown  by  the  scores. 


42  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

The  very  small  amount  of  iron  present  in  normal  butter  (averaging 
1.33  milligrams  per  kilo  for  10  samples  from  Troy  Creamery  Co.) 
necessitated  the  use  of  a  very  delicate  method.  The  colorimetric 
method  for  iron  with  potassium  sulphocyanid  is,  according  to  Neu- 
mann,1 so  delicate  as  to  show  1  part  of  iron  in  1,600,000  parts  of  water. 
This  method  was  employed  for  the  determination  of  the  iron. 

METHOD  OF  ANALYSIS. 

The  method  of  analysis  used  is  as  follows:  About  500  grams  of 
butter  are  melted  and  the  clear  supernatant  fat  separated  from  the 
curd  solution.  At  first  the  fat  was  disregarded  and  only  the  curd 
solution  used  for  analysis.  Later  the  fat,  too,  was  analyzed  and 
found  to  take  up  approximately  10  per  cent  of  the  total  iron  in  the 
butter,  so  that  in  all  later  work  the  fat  and  curd  were  both  analyzed 
for  iron  content.  The  curd  solution  is  evaporated  almost  to  dryness, 
the  remaining  fat  burned  off,  and  then  ashed  at  a  low  heat.  The  fat, 
by  heating  to  its  ignition  point,  will  ignite  and  burn;  the  residue  con- 
taining some  small  pieces  of  curd  which  remained  in  the  fat  is  ashed. 
This  ash,  when  total  iron  is  wanted,  is  added  to  the  ashed  curd  and  the 
whole  extracted  with  hot  dilute  hydrochloric  acid  and  filtered  into 
a  graduated  300  cubic  centimeter  flask.  It  may  be  necessary  to 
extract  several  times,  and  perhaps  even  burn  the  filter  and  residue 
and  again  extract  in  order  to  be  sure  to  get  out  all  the  iron.  In 
order  to  avoid  any  contamination  with  iron,  this  process  of  ashing  is 
carried  out  in  platinum  dishes  and  a  platinum  spatula  used  in  stirring 
or  scraping  the  ash  from  the  dish.  These  separate  extracts  and 
washings  are  all  filtered  into  the  same  volumetric  flask  and  diluted 
to  300  cubic  centimeters  with  iron-free  distilled  water.  This  solution 
is  then  oxidized  to  have  all  iron  in  the  ferric  condition  and  an  aliquot 
portion  (from  0.5  to  10  cubic  centimeters,  depending  on  the  iron 
present  in  the  solution)  transferred  to  a  50  cubic  centimeter  Nessler 
tube.  Then  5  cubic  centimeters  of  a  10  per  cent  solution  of  potassium 
sulphocyanid  are  added  and  the  whole  diluted  to  50  cubic  centimeters 
with  distilled  water.  These  samples  are  then  compared  with  a  set  of 
standards  made  by  using  known  amounts  of  a  solution  containing 
0.0001  gram  iron  per  cubic  centimeter  and  made  up  to  50  cubic 
centimeters  in  the  same  way  as  the  unknown.  These  standards  fade 
on  standing,  so  they  should  be  compared  shortly  after  the  standards 
and  unknowns  are  made.  The  results  are  calculated  to  parts  per 
million  (milligrams  per  kilo). 

By  such  an  analysis  the  actual  amount  of  iron  in  the  butter  samples 
is  determined.     In  most  cases  the  iron  was  added  to  the  cream,  and 

i  Neumann,  B.  Die  Grenzen  der  Empflndlichkeit  verschiedener  Reactionen  auf  Metalle.  Chemiker- 
Zeitung,  vol.  20,  no.  79,  pp.  763-764.    Cothen,  Sept.  30, 1896. 


THE   EFFECT   OF   METALS   ON   BUTTER.  43 

although  the  conditions  were  kept  as  nearly  alike  as  possible,  still 
a  difference  in  the  consistency  of  the  butter  when  washed,  or  the 
thoroughness  and  length  of  the  draining  and  washing  would  make  a 
difference  in  the  amount  of  iron  retained  in  the  butter. 

Notwithstanding  the  fact  that  care  was  taken  to  get  the  best  possi- 
ble cream,  the  iron  content  can  not  be  expected  to  be  so  low  as  in 
cases  where  the  milk  is  drawn  into  glass  vessels  and  that  portion 
used  for  analysis.  The  normal  creams  for  about  34  samples  had 
an  average  iron  content  of  1.53  milligrams  per  kilo,  with  a  maximum 
of  3.8  and  a  minimum  of  0.45.  Only  6  samples  showed  over  2 
milligrams  per  kilo.  These  samples  were  taken,  some  at  Albert  Lea, 
Minn.,  some  at  Troy,  Pa.,  and  some  at  Washington,  D.  C. 

Relation  of  iron  in  butter  to  iron  in  the  cream. — Kooper,1  in  his 
investigations  found  that  by  washing  and  working  butter  it  would 
lose  some  of  its  iron  content.  In  this  work,  although  care  was  taken 
to  add  the  same  relative  amount  of  wash  water  and  to  work  and 
wash  the  butter  with  the  same  number  of  revolutions,  the  iron  found 
in  the  butter  did  not  show  any  definite  relation  to  the  amount  of  iron 
added  to  the  cream.  In  the  first  series  of  experiments  analyses  were 
made  of  the  cream,  butter,  buttermilk,  and  wash  water  in  an  effort 
to  determine  whether  there  was  any  definite  relation  between  the 
amount  of  metal  added  to  the  cream  and  that  found  in  the  butter, 
buttermilk,  and  wash  water,  but,  as  will  be  seen  by  the  following  table, 
there  was  no  uniformity  in  the  results.  The  analyses  were  made  on 
samples  of  about  500  grams  and  calculated  to  milligrams  per  kilo. 
The  total  amount  of  butter,  buttermilk,  and  wash  water  were  not 
weighed  accurately,  and  so  the  total  weights  of  iron  in  the  table  are 
only  approximated.  In  the  following  results  the  total  approximated 
milligrams  of  iron  in  butter,  buttermilk,  and  wash  water  have  been 
shown,  as  well  as  the  percentage  return  of  iron  in  each  of  these.  A 
percentage  comparison,  however,  unless  based  on  the  same  amount 
of  cream  (of  same  fat  content),  butter,  buttermilk,  and  wash  water ; 
and  to  the  cream  of  which  the  same  amount  of  iron  has  been  added 
in  each  case,  really  does  not  enable  us  to  draw  any  definite  conclu- 
sions. If  the  amount  of  iron  added  were  small,  a  small  variation  in 
the  amount  returned  would  make  a  very  much  larger  percentage 
difference  than  if  the  same  variation  were  shown  on  a  larger  addition 
of  iron.  This  would  be  especially  marked  on  controls,  the  cream  of 
which  would  have  from  1  to  2  milligrams  of  iron  per  kilo,  while  the 
butter  in  controls,  however  carefully  made,  would  show  as  much  or  in 
most  cases  more  than  the  cream,  to  say  nothing  of  the  amount  of  iron 
found  in  the  buttermilk  and  wash  water.     The  possible  error  of 

» Kooper,  W.  D.    Loc.cit. 


44 


CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 


sampling  when  working  on  these  small  amounts  also  tends  to  mini- 
mize the  value  of  the  percentage  relations. 

Following  are  some  of  the  results  as  found,  the  milligrams  per  kilo 
being  accurate  but  the  total  weights  of  iron  only  approximated : 


Table  9. 


-Relation  between  iron  added  to  cream  and  the  ironjound  in  butter,  buttermilk, 
and  wash  water. 


Butter 
No. 

Cream. 

Butter. 

Buttermilk. 

Wash  water. 

Iron 

Total 

Iron 

Total 

Total 

Per- 
centage 

Total 

Per- 
centage 

of 
added 

iron. 

Iron 

Total 

Per- 
centage 
of  added 

iron. 

added. 

iron 
added. 

found. 

iron. 

iron 
found. 

of  added 
iron. 

iron. 

found. 

iron. 

Mas. 
per  kilo. 

Mgs. 

Per 

Mas. 
per  kilo. 

Per 

Mas. 
per  kilo. 

Mgs. 

per  kilo. 

Mgs. 

cent. 

Mgs. 

cent. 

Mgs. 

Per  cent. 

8 

1,000 

2,736.0 

180.90 

172 

6.3 

1,189.00 

2,310.0 

84.40 

17.70 

35 

1.30 

13 

500 

1,310.0 

84.00 

77 

5.9 

510. 20 

962.0 

73.40 

36.40 

73 

5.60 

19 

200 

9,613.0 

29.40 

402 

4.2 

69.60 

2,554.0 

26.57 

92.60 

4,222 

42.92 

27 

100 

4,808.0 

11.40 

140 

2.9 

61.20 

2,252.0 

46.85 

2.90 

137 

2.90 

38 

50 

2,296.0 

4.29 

66 

2.8 

24.10 

914.0 

39.80 

15.60 

711 

30.97 

81 

50 

2,269.0 

15.60 

212 

9.3 

39.40 

1,253.0 

55.22 

1.20- 

27 

1.20 

51 

20 

862.0 

7.78 

85 

9.9 

8.89 

304.0 

35.27 

.40 

10 

1.20 

155 

20 

862.0 

8.60 

112 

13.0 

18.90 

605.0 

70.20 

7.20 

164 

23.95 

43 

55 

(s) 

40.0 

3.03 

33 

82.5 

2.17 

58.7 

146.30 

1.44 

•  33 

82.50 

85 

103.2 

2.22 

39 

37.8 

1.73 

75.0 

72.70 

.97 

22 

21.30 

1  Iron  in  contact  twenty  minutes.  3  Control.    Found  1  milligram  per  kilo. 

3  Control.    Found  1.98  milligrams  per  kilo. 

Although  from  uiese  results  no  definite  relationship  can  be  stated 
between  the  amount  of  iron  added  to  the  cream  and  the  amount  of 
iron  found  in  the  butter,  buttermilk,  and  wash  water,  yet  in  a  general 
way  it  might  be  stated  that  a  relatively  small  part  of  the  iron  goes 
into  the  butter  as  compared  with  the  buttermilk,  which  seems  to  take 
most  of  the  iron,  and  in  which  the  presence  of  a  flavor  due  to  the  iron 
was  most  noticeable. 

Distribution  of  iron  between  fat  and  curd  solution. — When  this  work 
was  first  started  it  was  thought  that  the  amounts  of  iron  taken  up  by 
the  fat  of  the  butter  were  negligible,  and  so  in  the  analyses  of  butter 
the  curd  solution  only  was  used  for  analysis.  Later  analyses  were 
made  of  the  fat  as  well  as  the  curd  solution  on  22  samples  of  butter 
made  at  Troy,  Pa.,  during  the  summer  of  1911,  with  the  following 
results,  basing  the  fat  content  in  butter  at  80  per  cent,  since  all  the 
fat  was  not  separated  from  the  curd  solution  and  the  fat  contained 
small  particles  of  curd.  The  fat  was  not  filtered,  and  so  in  some 
instances  contained  small  particles  of  curd,  which  may  account  for  a 
variaDce  in  iron  content  of  the  fat. 


THE   EFFECT   OF    METALS   ON    BUTTER. 
Table  10. — Iron  content  oj  butter,  curd,  and  Jot. 


45 


Iron 
added  to 

Total 

Percent- 

Percent- 

Butter 

iron 

age  of 

age  of 

Iron  in 

Iron  in 

No. 

found  in 

total  iron 

total  iron 

fat. 

curd. 

cream. 

butter. 

in  fat. 

in  curd. 

Mgs.  per 

Mgs.  per 

Mas.  per 
kilo. 

Mas.  per 
kilo. 

kilo. 

kilo. 

Per  cent. 

Per  cent. 

120 

0 

6.64 

7.90 

92.10 

0.66 

30.59 

122 

20 

25.60 

22.70 

87.30 

7.27 

98.80 

125 

0 

6.92 

9.40 

90.60 

.82 

31.32 

127 

10 

10.99 

3.25 

96.75 

.45 

53.18 

132 

0 

9.11 

4.20 

95.80 

.48 

43.64 

134 

5 

7.20 

10.50 

89.50 

.95 

32.25 

140 

0 

8.24 

8.40 

91.60 

.87 

41.38 

142 

2 

7.40 

12.00 

88.00 

1.11 

32.55 

148 

0 

7.56 

12.00 

88.00 

1.13 

33.27 

1.50 

1 

8.79 

10.70 

89.30 

1.18 

39.28 

157 

0 

5.45 

15.50 

84.50 

1.06 

23.01 

159 

0 

5.09 

32.40 

67.60 

2.06 

17.21 

164 

0 

4.62 

27.50 

72.50 

1.59 

16.75 

166 

0 

5.71 

17.90 

82.10 

1.28 

23.43 

171 

0 

4.04 

19.60 

80.40 

.99 

16.22 

173 

0 

7.76 

13.10 

86.90 

1.27 

33.70 

193 

0 

4.78 

9.39 

90.61 

.56 

21.73 

194 

0) 

6.33 

7.44 

92.56 

.59 

29.29 

201 

0 

6.77 

8.03 

91.97 

.68 

31.11 

203 

(») 

6.93 

10.00 

90.00 

.89 

31.21 

205 

0 

5.18 

8.91 

91.19 

.58 

23.61 

207 

(2) 

9.11 

7.69 

92.31 

.88 

42.07 

1  Rusted  can. 


2  Iron  strip. 


In  this  table,  as  in  any  table  of  this  kind,  the  percentage  relation 
is  relatively  unimportant.  The  question  of  the  actual  amount  of 
iron  found  in  the  fat  is  of  much  greater  importance.  An  average 
of  the  control  butters,  of  which  there  are  14  in  the  above  table,  will 
show  a  content  of  1  milligram  of  iron  per  kilo  of  butter  fat,  a  very 
small  amount  that  could  be  disregarded  in  most  cases.  In  normal 
butters  made  under  proper  conditions  where  the  total  iron  content 
is  about  1.3  milligrams  per  kilo,  even  a  difference  of  0.5  milligram 
of  iron  in  a  pound  of  butter  would  be  noticeable  on  a  comparative 
basis  between  the  fat  and  curd  content,  but  when  total  iron  is  being 
considered  so  small  an  amount  as  0.5  milligram  of  iron  could  easily 
be  discarded  in  the  calculation. 

THE    INFLUENCE   OF   IRON   ON   FLAVOR. 

The  ideal  condition  for  the  solution  of  this  problem  would  of 
course  be  to  have  butter  made  absolutely  free  from  iron  while  known 
amounts  of  iron,  or  any  other  metal  as  the  case  might  be,  could  be 
added  to  a  portion  of  the  cream  used  in  making  this  metal-free 
butter.  This  could  be  done  only  by  drawing  the  milk  samples 
directly  into  glass  vessels,  skimming  by  hand,  and  ripening  and 
churning  in  small  quantities  in  glass.  This  method  of  procedure 
seemed  impracticable,  since  enough  butter  could  not  be  made  to 
satisfy  all  the  requirements  of  the  experiment.  The  first  few  sam- 
ples were,  however,  churned  in  a  tall  glas3  jar  by  shaking  by  hand, 
but  proved  unsatisfactory  in  that  only  a  small  amount  could  be 


46 


CHANGE   IN   FLAVOR  OF   STORAGE  BUTTER. 


made  and  the  butter  was  not  very  good.  The  best  thing  to  be 
done  under  the  circumstances  was  to  control  conditions  as  carefully 
as  possible  and  avoid  any  undue  contact  with  iron  during  the  whole 
process  of  butter  making.  The  method  of  butter  making  has  been 
described.  The  butters  were  scored  for  the  first  time  in  most  cases 
very  shortly  after  making,  being  kept  in  cold  storage  until  scored. 
It  will  be  noticed  that  the  butters  made  at  Albert  Lea,  Mirm.,  were 
scored  by  the  Fox  River  Butter  Co.  and  those  made  at  Troy,  Pa., 
or  Washington,  D.  C,  were  scored  by  Mr.  Fryhofer  and  Mr.  Smarzo, 
of  New  York  City.  For  this  reason  these  scores  are  not  comparable, 
as  in  our  experience  there  is  considerable  variance  between  the 
scores  and  methods  of  scoring  even  among  professional  butter 
scorers.  Following  is  a  table  showing  the  butter  scores.  In  every 
case  the  butter  to  which  metal  has  been  added  is  followed  by  a  con- 
trol  butter,  made  at   the  same  time  under  the   same   conditions. 

Table  11. — Influence  oj  iron  on  flavor  of  butter. 


Data  on  ripened,  pasteurized  cream. 

Butter. 

Butter 
No. 

Iron 

Iron 

Dura- 

Acidity 

Iron 

Age  of 

content 

added  as 

tion  of 

at  time 

found 

butter 

Score. 

Remarks. 

normal 

ferrous 

Con- 

of churn- 

in 

when 

in  cream. 

sulphate. 

tact. 

ing. 

butter. 

scored. 

Per  cent 

Mgs.  per 
kilo. 

Mgs.  per 
kilo. 

of  lactic 

Mgs.per 

Hours. 

acid. 

kilo. 

Days. 

U9 

200 

22 

29.4 

7 
187 

85.0 
85.0 

Very  fishy. 

Do. 

123 

10.90 

0 

0.53 

8.3 

7 
187 

92.5 
85.0 

Coarse,  unclean. 
Very  fishy. 

27 

3.84 

100 

23 

.77 

11.4 

11 
184 

92.0 
85.0 

Oily. 
Very  fishy. 

31 

3.84 

0 

.65 

2.59 

11 
184 

93.5 
85.0 

Do. 

39 

.40 

50 

23 

.73 

4.29 

14 

179 

86.0 
85.0 

Fishy. 
Very  fishv. 

135 

.40 

0 

.73 

1.70 

14 
179 

87.0 
85.0 

Fishy. 
Very  fishy. 
Slightly  fishy. 

1-47 

.99 

25 

23 

.53 

26.5 

9 

87.0 

175 

85.0 

Very  fishy. 

M3 

1.00 

0 

.51 

3.03 

9 
175 

92.0 
86.0 

Coarse. 
Very  fishy. 

181 

2.76 

50 

22 

.60 

15.6 

16 

160 

93.5 
88.0 

Clean. 

Oily,  unclean. 

177 

2.76 

0 

.62 

2.49 

16 

160 

94.0 
88.0 

Fishy. 

122 

2.93 

20 

21 

.57 

25.6 

3 
44 
116 

86.0 
85.0 
82.0 

Very  oily;  very  fishy. 

Do. 
Rank,  oily,  and  fishy. 

120 

2.90 

0 

.58 

6.64 

3 
44 

93.0 
84.5 

Clean. 

Very  fishy. 

High  acid,  clean,  rank  fishy. 

116 

80.0 

127 

2.11 

10 

23 

.49 

10.99 

5 
42 
114 

84.0 
85.0 
84.0 

Oily,  metallic,  unclean,  rancid. 
Oily,  metallic,  very  salvy. 
Very  oily,  metallic. 
Oily,    shghtly    metallic,    un- 
clean. 

125 

2.48 

0 

.48 

6.92 

5 

88.0 

42 

85.0 

Oily,  metallic. 

114 

84.0 

Very  oily,  metallic. 

134 

1.64 

5 

21* 

.59 

7.20 

3 
39 
111 

91.5 
86.5 
83.0 

Metallic,  unclean. 
Metallic,  stale. 
Very  fishy. 

132 

1.64 

0 

.55 

9.11 

3 
39 
111 

94.0 
85.5 
82.0 

Very  clean. 
Fishy. 

Very   high  acid,   clean,   very 
fishy. 

•  Butter  made  at  Albert  Lea,  Minn.    Scored  by  Fox  River  Butter  Co.    All  other  butters  made  at 
Troy,  Pa. 


THE   EFFECT   OF   METALS   ON   BUTTER. 
Table  11. — Influence  oj  iron  on  flavor  oj  butter — Continued. 


47 


Butter 
No. 


Data  on  ripened,  pasteurized  cream. 


Iron 

content 

normal 

in  cream. 


Iron 

added  as 

ferrous 

sulphate. 


Dura- 
tion of 
con- 
tac. 


Acidity 
at  time 
of  churn- 
ing. 


Iron 
found 


butter. 


Butter. 


Age  of 
butter 
when 
scored. 


Score. 


Remarks. 


142 
140 
150 

148 

193 
192 
223 

222 

225 

224 

227 

226 

229 

.228 

231 

230 


Mgs.  per 
kilo. 
1.17 


1.17 
.45 
.74 


2.99 
2.73 


Mgs.  per 
kilo. 
2 


0 
1 

0 

20 
0 

2  10 

0 
»5 

0 
2  2.5 

0 

22 

0 

21 

0 


Hours. 
17 


Per  cent 
of  lactic 
acid. 
.59 


20J 


21 


UH 


203 


.552 

.66 

.55 

.60 

.54 

.44 

.42 

.46 

.52 


Mgs. per 
kilo. 
7.40 


8.24 

8.79 

7.56 

5.80 
3.41 
7.37 

3.39 

5.4 

2.15 

3.59 

2.85 

1.61 

1.89 

1.63 

2.16 


Days. 

2 
47 
105 

2 
47 
105 

6 
45 
103 

6 
45 
103 
11 
128 
11 
128 

4 
29 
61 

4 
29 
61 

4 
27 
59 

4 
27 
59 

4 
25 
57 

4 
25 
57 

7 
22 
54 

7 
22 
54 

5 
20 
52 

5 
20 
52 


91.0 
87.0 
88.0 
94.5 
88.0 
83.0 
86.0 
86.0 
85.0 
89.0 
87.0 
86.0 
86.0 
86.0 
91.5 
91.0 
84.0 
84.0 
84.0 
86.0 
86.0 
86.0 
88.0 
86.0 
84.0 
90.0 
87.0 
87.0 
87.0 
87.0 
85.0 
93.0 
93.0 
89.0 
88.0 
87.0 
85.0 
92.0 
92.0 
88.0 
94.0 
93.0 
86.0 
92.0 
92.0 
88.0 


Slightly  metallic. 

Very  metallic. 

Oily. 

Slightly  metallic  aroma,  clean. 

Metallic. 

Very  fishy,  clean,  low  acid. 

Very  oily  and  fishy. 

Oily  and  fishy. 

Do. 
Milky 

Slightly  fishy. 
Fishy,  clean,  high  acid. 
Oily,  very  fishy. 
Fishy. 


Unclean,  stale,  oily. 
Unclean,  metallic,  gritty. 
Rank,  metallic. 
Stale,  oily. 
Very  oily,  metallic. 
Oily,  metallic. 

Do. 
Very  oily. 

Very  metallic  and  fishy. 
Slightly  metallic. 
Unclean,  oily. 
Oily,  metallic. 
Tainted,  very  oily. 
Oily. 

Very  oily,  metallic. 
Good  butter. 

Old  flavor. 
Very  metallic. 
Oily,  metallic. 
Very  metallic. 
Somewhat  oily,  coarse. 

Oily,  trifle  fishy. 
Good,  clean,  creamy. 

Very  fishy. 
Somewhat  oily,  coarse. 


Little  fishy. 


i  Butter  made  at  Albert  Lea,  Minn.    Scored  by  Fox  River  Butter  Co.    All  other  butters  made  at 
Troy,  Pa. 
'  Iron  added  as  lactate. 

In  Table  11  it  will  be  noticed  that  in  every  instance  on  the  first 
scoring  the  butters  to  which  iron  had  been  added  scored  lower  than 
their  controls.  This  holds  in  most  cases  on  the  second  and  third 
scoring,  the  most  noticeable  feature  being  that  the  butters  to  which 
iron  has  been  added  show  the  deterioration  much  faster  than  the 
control  butters.  After  the  butters  have  deteriorated  to  a  score  of 
85  or  lower,  the  butter  is  so  poor  that  a  difference  of  a  point  or  two 
in  the  score  really  does  not  indicate  a  very  great  difference  in  the 
quality  of  the  butter.  A  great  many  of  the  butters  became  fishy, 
and  where  both  were  not  scored  fishy  at  the  same  time  it  will  be 
noticed  that  the  control  butter  was  the  last  to  become  "fishy,"  though 


48 


CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 


in  one  or  two  instances  the  control  was  scored  "fishy,"  while  the 
other  butter  was  not  marked  fishy  at  all.  A  very  noticeable  feature 
about  these  butters  is  the  production  of  a  very  oily  flavor.  This 
was  present  in  most  amples  that  were  marked  fishy  and  seems  to  be 
a  stepping  stone  to  the  fishy  flavor. 

THE  INFLUENCE  OF  COPPER  ON  FLAVOR. 

In  the  work  on  copper  only  the  physical  changes  will  be  con- 
sidered, as  considerable  difficulty  was  experienced  in  making  accurate 
determinations  of  the  very  small  amounts  of  copper  present  in  the 
butter. 

These  butters  were  made  in  exactly  the  same  way  as  the  butters 
showing  the  influence  of  iron.  The  cream  was  all  very  carefully 
handled  to  avoid  contact  with  copper,  and  divided  into  two  portions, 
which  were  treated  in  exactly  the  same  manner,  excepting  that  to 
one-half  of  the  cream,  copper  in  the  form  of  a  solution  of  copper 
sulfate  or  lactate  was  added  in  amounts  varying  from  one-half  to 
20  milligrams  per  kilo  of  cream.  The  cream  was  ripened  and  churned 
and  the  butter  stored  in  glass  jars  without  metal  parts,  or  in  ash 
tubs,  in  1912,  and  scored  at  intervals.  The  changes  in  the  butter 
are  shown  in  the  following  tables: 

Table  12. — Influence  of  copper  on  butter. 


Data  on  ripened  and  pasteur- 
ized cream. 

Butter. 

Butter 
No. 

Copper 
added 

as  sul- 
fate. 

Duration 
of  con- 
tact. 

Acidity 
at  churn- 
ing. 

Age  on 
scoring. 

Score. 

Remarks. 

189 
i  85 

Mas.  -per 
kilo. 
20 

0 

4 

0 

2 

0 

1 

0 
<5.0 

0 
4  2. 5 

Hours. 
19 

Per  cent 
of  lactic 
acid. 
0.51 

.60 

.60 

.60 

.56 

.52 

.51 

.50 

.36 

.385 

.415 

Days. 

14 

158 

14 

158 

5 

38 
96 

5 
38 
96 

2 
40 
98 

2 
40 
98 

4 
42 
100 

4 
42 
WO 

3 
18 
50 

3 

18 
50 

2 
15 
47 

90.0 
85.0 
92.5 
86.0 
85.0 
85.0 
80.0 
91.0 
90.0 
86.0 
87.0 
84.0 
80.0 
93.0 
87.0 
87.0 
85.0 
84.0 
80.0 
91.0 
86.0 
82.0 
86.0 
84.0 
84.0 
92.0 
89.0 
89.0 
88.0 
87.0 
84.0 

Veryfishy. 
Oily. 

*173 
»171 

22 

Very  fishy. 

Oily,  fishy,  unclean. 

Oily,  fishy. 

Rank,  fishy,  clean,  high  acid. 

Oily. 

»166 

Slight  metallic. 
Fishy,  clean,  low  acid. 
Oily,  unclean,  rancid. 

J  164 

Very  oily,  fishy. 

Rank,  oily  and  fishy,  unclean,  low  acid. 

»159 

- 

Oily,  metallic. 

Oily. 

Oily,  metallic,  stale. 

a  157 

Very  oily  and  fishy. 

Rank,  fishy,  unclean,  low  acid. 

»235 

'234 

19 

Very  oily,  fishy. 

Very  oily,  fishy,  clean,  high  acid. 

Very  oily  and  metallic. 

Oily  and  fishy. 

Rank,  fishy. 

»237 

19 

Oily. 

Old  flavor. 
Oily  and  metallic. 
Unclean,  oily. 
Rank,  fishy. 

1  Butter  made  at  Albert  Lea,  Minn.,  1910. 
•  Butter  made  at  Troy,  Pa.,  1911. 


a  Butter  made  at  Troy,  Pa.,  1912. 

4  Copper  added  in  form  of  copper  lactate. 


THE  EFFECT   OP   METALS   ON  BUTTER. 
Table  12. — Influence  of  copper  on  butter — Continued. 


49 


Data  on  ripened  and  pasteur- 
ized cream. 

Butter. 

Butter 
No. 

Copper 
added 
as  sul- 
fate. 

Duration 
of  con- 
tact. 

Acidity 
at  churn- 
ing. 

Age  on 
scoring. 

Score. 

Remarks. 

1236 

Mgs.  per 
kilo. 
0 

"1.5 

0 

2  1.0 

0 

Hours. 

Per  cent 
oflactic 
acid. 
.58 

.376 

.355 

.40 
.34 

Days. 

2 
15 
47 

2 
13 
45 

2 
13 
45 

3 
43 

3 
43 

92.0 
91.0 
86.0 
92.0 
92.0 
86.0 
94.0 
94.0 
93.0 
88.0 
87.0 
91.0 
91.0 

Trifle  coarse,  slightly  tainted. 

1239 
i  238 

181 

Slight  unclean. 
Tallowy  flavor. 
Slight  oily  and  metallic. 
Slight  oily. 
Metallic  and  fishy. 

1241 
i  240 

21 

Clean. 

Very  oily  and  metallic. 

Metallic. 

Trifle  metallic. 

Old  flavor. 

Butter  made  at  Troy,  Pa.,  1912. 


:  Copper  added  in  form  of  copper  lactate. 


From  Table  12  it  will  be  seen  that  in,  every  instance  the  scores  on 
the  control  butters  were  better  than  the  scores  on  the  butter  made 
from  cream  to  which  copper  had  been  added,  even  in  the  small 
amount  of  1  milligram  of  copper  per  kilo  of  cream. 

Unfortunately,  the  "Remarks"  on  the  butter  do  not  show  any 
definite  characteristics  that  can  be  attributed  to  the  copper.  The 
control  butters,  too,  show  deterioration  in  storage,  though  they  seem 
to  keep  better  than  the  butters  made  from  cream  to  which  the  copper 
was  added.  On  the  second  scoring  after  40  days  in  storage  most  of 
the  butter  to  which  copper  had  been  added  showed  a  fishy  flavor 
and  after  three  months  a  very  decided  rank,  fishy  flavor  that  was 
unmistakable,  and  was  called  by  scorers  decided  mackerel  flavor. 
These  butters  were  all  made  during  the  summer  months. 

An  experiment  to  show  the  effect  of  having  the  cream  stand  in 
contact  with  a  small  surface  of  copper  for  a  long  time  was  made  by 
having  a  vat  of  cream  ripened  in  contact  with  two  sheets  of  bright 
copper  each  2  by  6  inches.  The  sheets  were  placed  on  edge  in  the 
bottom  of  the  vat  during  the  process  of  ripening.  The  result  of  this 
is  shown  in  the  following  table: 

Table  13. — Effect  oj  copper  on  flavor  of  butter. 


Butter 
No. 

Cream. 

Duration 
of  con- 
tact. 

Age. 

Score. 

Normal 
acidity. 

Churning 
acidity. 

Remarks. 

256 

Per  cent. 
0.17 

.17 

Per  cent. 
0.355 

.37 

Hours. 

Days. 

1 
31 

1 
31 

93 
91 
91 
88 

257 

21 

Trifle  old  flavor. 
Trifle  oily. 
Metallic. 

50 


CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 


The  difference  in  flavor  is  marked  in  the  fresh  butter  but  more 
marked  in  storage  butter. 

Another  experiment  to  show  the  effect  on  the  flavor  of  the  butter, 
by  having  the  cream  come  in  contact  with  a  large  surface  of  copper 
for  a  short  time,  was  made  by  pasteurizing  the  cream  in  two  parts. 
One  part  was  pasteurized  in  a  No.  3  Peerless  pasteurizer,  the  copper 
lining  being  completely  covered  with  tin.  The  second  part  was 
pasteurized  in  a  No.  5  Peerless  pasteurizer,  the  tin  coating  of  which 
had  been  worn  off  by  continued  usage,  leaving  the  cream  exposed 
to  a  surface  of  copper  during  the  process  of  pasteurization.  The 
duration  of  contact  with  the  copper  was  only  a  few  seconds,  though 
the  surface  was  quite  large.  The  temperature  of  pasteurization  was 
about  60°  C. 

The  results  of  this  experiment  are  found  in  the  following  table: 

Table  14. — Comparison  of  the  effect  oj  tin  and  copper  on  the  flavor  oj  butter. 


Acidity 
of  cream 
before 
pasteur- 
ization. 

Temper- 

Acidity 

Butter 
No. 

Pasteur- 
izer. 

ature  of 

pasteur- 
ization. 

at 

churn- 
ing. 

Age. 

Score. 

Remarks. 

Per  cent. 

°  C. 

Per  cent. 

Days. 

245 

Tin 

0.18 

60 

0.55 

2 

39 

91 

92 

Slight  oily. 
Good. 

246 

Copper. . 

.18 

60 

.50 

2 
39 

87 
85 

Oily,  stale,  metallic. 
Very  fishy. 
Slight  oily. 

252 

Tin 

.19 

60 

.52 

1 

92 

33 

89 

Oily. 

253 

Copper. . 

.19 

60 

.47 

1 

33 

89 
84 

Do. 
Very  fishy. 

These  two  experiments  show  very  plainly  the  deteriorating  effect 
of  poorly  tinned  pasteurizers,  for  aside  from  this  all  other  conditions 
were  exactly  alike  during  the  complete  process  of  butter  manufac- 
ture. Considering  the  short  duration  of  contact,  the  change. in  the 
flavor  of  the  butter  even  when  fresh  is  very  marked.  The  effect  of 
copper  even  in  small  amounts  seems  to  cause  more  marked  changes 
in  butter  flavor  than  iron,  with  a  marked  tendency  toward  a  fishy 
flavor  in  storage. 

CONTAMINATION   OF   CREAM    WITH    IRON    FROM    CONTAINERS. 

For  this  experiment  a  lot  of  cream  was  divided  into  two  portions, 
one  portion  being  held  in  a  clean  can  and  the  other  in  a  can  which 
showed  many  rust  spots.  This  was  the  most  rusted  of  all  the  cans 
that  were  used  for  the  handling  of  cream  at  the  Troy  Creamery  Co., 
of  Troy,  Pa.  However,  this  showed  only  small  spots  of  rust  on  the 
bottom  of  the  can.  This  raw  cream  was  ripened  without  the  addition 
of  a  starter  at  room  temperature.  The  temperature,  acidity,  and 
iron  content  are  as  follows: 


THE   EFFECT   OF   METALS   ON   BUTTER. 
Table  15. —  The  absorption  oj  ironjrom  rusty  can. 


51 


Clean  can 

Rusted  can. 

Duration 
of 

contact. 

Temper- 
ature. 

Acidity. 

Iron 
found. 

Temper- 
ature. 

Acidity. 

Iron 
found. 

Hours. 

'  F. 

Per  cent. 

Mgs.  p.  kilo. 

°  F. 

Per  cent. 

Mgs.  p.  kilo. 

0 

66 

0.114 

1.045 

66 

0.114 

1.045 

2 

67 

.101 

1.031 

67 

.120 

1.132 

4 

67 

.146 

1.539 

67 

.158 

1.772 

6 

67 

.177 

1.028 

67 

.176 

1.265 

8 

68 

.202 

1.677 

68 

.212 

1.129 

14 

66 

.412 

.792 

66 

.422 

1.627 

21 

66 

.561 

1.082 

66 

.561 

1.459 

26 

64 

.581 

.951 

56 

.568 

1.187 

32 

52 

.634 

1.082 

57 

.624 

1.257 

48 

44 

.632 

-1.185 

57 

.638 

2.381 

Butter  m 

ade  from  a 

jove  cream 

Butter  made  from  above  cream 

4.78. 

6.33. 

Another  experiment  on  the  same  basis  but  without  the  acidity  of 
the  cream  at  the  various  intervals  gave  the  following  results,  from  a 
cream  having  a  normal  iron  content  of  0.9  milligram  per  kilo  and  an 
acidity  of  0.16  per  cent  lactic  acid. 

Table  16. — Effect  oj  rusty  can  on  the  flavor  oj  butter. 


Butter. 

Iron  in 
butter. 

Cream 
contact 
in  cans. 

Iron 

content  in 

ripened 

cream. 

Churning 

Butter. 

Remarks. 

No. 

acidity. 

Age. 

Score. 

259 
260 

Mgs.  p.  kilo. 
1.37 

1.60 

Hours. 
15 

15 

Mgs.  p.  kilo. 
4.31 

4.35 

Per  cent. 
0.51 

.49 

Days. 
19 
29 
19 
29 

92 

89 
88 
86 

Slight  oily. 
Trifle  metallic,  oily. 
Oily,  metallic. 
Unclean,  oily. 

From  Table  15,  it  is  seen,  as  would  be  expected,  that  the  acidity 
of  cream  increases  with  the  length  of  time  it  is  held,  though  there  is 
no  material  difference  in  the  rate  of  increase  between  the  two  samples. 
The  cream  in  the  rusted  can  does  not  show  any  marked  increase  in 
iron  content  over  that  in  the  clean  can.  The  difficulty  in  getting  an 
accurate  sample  of  the  cream  after  standing  long  enough  to  thicken 
may  account  for  the  variation  from  what  might  have  been  expected. 
At  the  end  of  48  hours  the  cream  from  the  rusted  can  showed  an  iron 
content  of  1.2  parts  per  1,000,000  more  than  the  cream  from  the 
clean  can.  The  butters  made  from  these  creams  checked  very 
closely,  as  the  butter  made  from  the  cream  held  48  hours  in  the  rusted 
can  showed  1.55  parts  per  1,000,000  more  than  the  butter  made  from 
the  cream  held  48  hours  in  a  clean  can. 

In  another  instance  to  find  whether  cream  on  standing  in  contact 
with  iron  rust  would  take  up  iron,  a  lot  of  cream  was  divided  into 
four  parts,  each  placed  in  a  clean  can,  and  a  strip  of  rusted  iron  tape 
one-half  inch  wide  and  24  inches  long  (giving  a  surface  of  24  square 


52 


CHANGE   IN   FLAVOR  OF   STORAGE  BUTTER. 


inches)  was  placed  in  two  of  these  cans.  Two  cans,  one  with  and 
one  without  the  iron,  were  ripened  at  room  temperature  for  21  hours, 
and  two  cans,  one  with  and  one  without  iron,  were  held  in  cold 
storage  at  32°  F.  for  21  hours.  Butter  was  made  from  each  of  these 
four  samples  of  cream.  There  was  no  starter  added  to  this  cream. 
The  cream  from  which  these  four  lots  were  taken  had  acidity  of  .223 
and  iron  content  of  1.25  milligrams  per  kilo. 

Table  17. — Data  on  cream  ripened  in  contact  with  rusty  iron. 


No. 

Cream. 

Butter. 

Temper- 
ature. 

Acidity. 

Iron  con- 
tent. 

Butter 
No. 

Iron  con- 
tent. 

Score. 

1 

12 
3 

i  4 

'F. 
32 
32 
66 
66 

0.385 
.368 
.655 
.670 

Mas.  per 

kilo. 

1.53 

1.81 

.90 

11.32 

1 
2 
3 
4 

M  as.  per 
kilo. 
6.77 
6.93 
5.18 
9.11 

94 
91 
94 
91 

' :  In  contact  with  iron  tape. 

From  this  table,  although  it  does  not  show  the  acidity  and  iron 
content  at  intervals  as  in  Table  15,  still  it  shows  here  that  with 
the  development  of  acidity  the  iron  is  taken  up  by  the  cream. 
This  is  very  marked  in  cream  and  butter  No.  4,  the  iron  content 
in  both  being  very  high  as  compared  with  their  respective  controls. 
The  effect  of  the  iron  is  also  very  marked  in  the  butter  scores,  the 
controls  (Nos.  1  and  3)  scoring  three  points  higher  than  the  ones  to 
which  the  iron  had  been  added. 

In  the  first  experiment  Mr.  Larson,  superintendent  of  the  Troy 
Creamery  Co.,  said  he  could  taste  the  iron  in  the  cream  after  32 
hours'  contact,  and  in  the  second  experiment  the  cream  in  contact 
with  iron  ripened  at  room  temperature  showed  a  bitter  metallic 
taste. 

In  every  case  the  butter  made  from  cream  which  had  stood  in 
contact  with  iron  rust  showed  a  peculiar  taste  and  was  easily  picked 
out  from  a  lot  of  samples.  The  taste  was,  however,  most  noticeable 
in  the  buttermilk,  to  which  it  gave  a  decided  metallic  taste. 

In  Table  15  the  difference  in  the  amounts  of  iron  found  in  the 
cream  are  very  small,  in  only  one  case  being  more  than  1  milligram 
of  iron  per  kilo  of  cream.  Taking  into  consideration  the  possibility 
of  error  in  sampling  a  very  thick  cream,  these  small  differences  do 
not  seem  to  be  enough  to  warrant  any  definite  conclusions  as  to  the 
absorption  of  iron  by  the  cream.  It  will  be  noticed  in  Table  1 1  that 
all  the  control  butters  made  by  us  at  Troy,  Pa.,  have  a  very  high 
iron  content,  as  compared  with  control  butters  made  by  us  at  Albert 


THE   EFFECT   OF   METALS   ON  BUTTER.  53 

Lea  or  made  by  the  Troy  Creamery  Co.  at  Troy.  As  the  wash  water 
was  tested  and  the  cream  did  not  show  a  high  iron  content,  the 
churns  are  the  only  means  whereby  iron  might  be  introduced  into 
the  butter.  Although  these  churns  were  taken  apart  and  scraped, 
sandpapered,  and  thoroughly  cleaned  before  being  used  at  Troy,  it  is 
possible  that  there  were  some  rust  spots  on  the  iron  bolt  heads  and 
plates  coming  in  contact  with  the  cream,  and  that  the  iron  was 
attacked  at  these  points  by  the  high  acid  cream  and  the  iron  taken 
up  by  the  butter.  Unfortunately  the  butter  samples  were  not 
analyzed  at  the  time  the  butter  was  made,  otherwise  this  error 
would  have  been  noticed  in  the  chemical  results  and  corrected  before 
continuing  the  work.  The  high  iron  content  of  the  butters  made  at 
Troy,  Pa.,  seemed  to  show  conclusively  that  there  was  an  error  by 
contamination  at  some  step  in  the  process  of  butter  making.  The 
only  check  possible  on  the  butter  itself  was  by  analysis  of  the  butter 
made  by  the  Troy  Creamery  Co.  in  their  large  churn.  In  most  cases 
the  cream  for  the  experimental  butter  was  selected  from  the  best 
cream  brought  to  the  creamery,  while  the  cream  used  in  the  creamery 
proper  was  the  regular  run  of  cream  as  brought  in  by  the  farmers 
and  from  the  several  skimming  stations.  In  this  way  the  butter 
from  the  creamery  proper,  if  the  fault  were  in  the  cream,  should 
show  a  higher  iron  content  than  the  experimental  butter.  The 
cream  used  in  the  experimental  creamery  was  analyzed  for  iron  (in 
most  cases)  at  three  stages;  first,  raw  sweet  cream;  second,  immedi- 
ately after  pasteurizing  (to  determine  whether  there  was  any  iron 
exposed  in  the  pasteurizer  or  cooler),  and,  third,  after  ripening  in  the 
ripening  vats  to  determine  whether  there  was  any  iron  taken  up  in 
the  vats.  The  butter  was  then  analyzed,  thus  giving  the  last  stage  in 
the  process  of  butter  making. 

The  butter  made  in  1912  at  Troy,  Pa.,  was  churned  in  new  No.  5 
Bestov  box  churns,  and  worked  on  a  table  worker,  eliminating 
this  contamination  by  iron  in  the  churn.  The  butter  for  scoring 
was  packed  in  10-pound  ash  butter  tubs  instead  of  glass  jars  in  order 
to  avoid  injuring  the  body  of  the  butter  in  packing. 


54 


CHANGE   IN   FLAVOR   OF    STORAGE   BUTTER. 

Table  18. — Iron  content  oj  cream  and  controls. 
[Milligrams  per  kilo.] 


Butter. 

Raw 
cream. 

Pasteur- 
ized 
cream. 

Raw 
ripened 
cream. 

Pasteur- 
ized 
ripened 
cream. 

Curd  as 
100  per 
cent  of 
butter. 

Curd  as 
20  per 
cent  of 
butter. 

Fat  as 
80  per 
cent  of 
butter. 

120  E 
125  E 
132  E 
140  E 
148  E 
157  E 
164  E 
171  E 
201  E 
205E 

135  T 

136  T 
143  T 

151  T 

152  T 

160  T 

161  T 

167  T 

168  T 

195  T 

196  T 

197  T 

198  T 
'224 
'226 
•'228 
'230 
'234 
'236 
'238 
'240 
'246 
'247 
'248 
'252 
'253 
'256 
'257 
'259 
'263 
'264 

2.93 
2.57 
1.64 
1.17 
.74 
1.59 
1.63 
1.44 

6.12 
6.26 
8.73 
7.54 
6.65 
4.60 
3.35 
3.24 
6.22 
4.72 
1.12 
1.16 
1.25 
.96 
1.19 
.89 
1.21 
1 1. 55 
i  1. 31 
11.50 
»  1.79 
»    .99 
12.35 
2.15 
2.85 
1.89 
2.16 
2.04 
3.16 
3.50 
3.59 
1.95 
1.36 
1.14 
1.41 
1.41 
3.40 
6.63 
1.37 
2.69 
1.45 

30.59 

31.32 

43.64 

41.38 

33.27 

23.01 

16.75 

16.22 

31.11 

23.61 

5.59 

5.81 

6.26 

4.81 

5.97 

4.47 

6.05 

'7.77 

16.52 

17.51 

18.% 

i  4.96 

i  11.74 

0.66 
.82 
.48 
.87 
1.13 
1.06 
1.59 
.99 
.68 
.58 

2.11 
1.89 
1.19 
.45 
1.47 
1.46 
1.94 
1.25 
1.75 

2.48 
1.49 
1.12 
.55 

1.53 
.90 

! 

2.73 

.94 

2.04 

2.01 
1.14 

1.67 
1.67 

1.10 
.9 

4.31 
4.35 

E  Butter  made  in  experimental  creamerv,  Troy,  Pa.,  1911. 

T  Butter  made  by  Troy  Creamery  Co.,  1911. 

i  Curd  burned  in  porcelain. 

'  Butter  made  in  experimental  creamery,  Troy.  Pa.,  1912. 

Table  18  is  arranged  to  show  the  iron  content  found  in  the  cream 
and  butter  made  in  the  experimental  creamery  and  also,  for  compari- 
son, the  iron  content  of  the  butters  made  by  the  Troy  Creamery  Co. 
In  these  analyses  the  fat  was  disregarded  and  the  iron  found  in  the 
curd  solution  used  as  the  total  iron  in  the  butter.  It  will  be  noticed 
in  the  column,  "Fat  as  80  per  cent  of  the  butter"  that  the  iron 
content  averages  0.89  milligram  per  kilo.  As  the  usual  charge  of 
butter  was  500  grams,  the  total  iron  in  fat  would  be  less  than  0.4 
milligram  of  iron,  which  is  a  very  small  and  almost  negligible  quan- 
tity. 

Ten  samples  of  control  butter  made  in  the  experimental  churns 
gave  an  average  iron  content  of  5.74  milligrams  of  iron  per  kilo  of 
butter  using  the  curd  solution  only  and  calculating  as  butter,  while  13 
samples  of  butter  made  by  the  Troy  Creamery  Co.  in  their  large  churn 


THEOEETICAL   CONSIDERATIONS.  55 

during  the  same  period  showed  an  average  iron  content  of  1 .33  milli- 
grams of  iron  per  kilo  of  butter,  using  curd  solution  only  and  calcu- 
lating as  butter.  The  same  cream,  with  a  possible  advantage  in 
favor  of  the  experimental  cream,  the  same  salt,  and  the  wash  water 
from  the  same  source,  were  used  in  the  manufacture  of  both  the  experi- 
mental and  the  Troy  Creamery  Co.'s  butter,  so  that  the  only  point 
of  entry  for  the  increased  iron  content  in  the  experimental  butters 
was  in  the  churns.  This  might  possibly  account  for  the  deterioration 
in  the  control  butters  made  in  the  experimental  creamery  in  1911,  and 
although  these  results  detract  from  the  results  in  the  other  experiments, 
still  the  value  of  the  evidence  to  show  that  iron  may  be  taken  up  in 
the  churn  is  of  great  importance  to  the  butter  maker.  Rusty  bolt- 
heads,  plates,  or  other  castings  should  be  carefully  guarded  against 
by  the  butter  maker. 

THEORETICAL  CONSIDERATIONS. 

Having  ascertained  definitely,  partly  from  the  empirical  observa- 
tions of  others,  and  partly  from  the  experimental  data  obtained  on 
butter  containing  added  iron,  that  iron  itself  lowers  the  keeping 
quality  of  butter,  it  was  desirable  to  find  out  how  iron  affects  butter. 

That  the  iron  acts  catalytically  in  an  oxidative  reaction  at  once 
suggests  itself.  As  has  been  shown,  butter  made  by  the  usual 
methods  contains  gases  in  the  proportion  of  approximately  10  cubic 
centimeters  in  100  grams  of  butter.  It  seems  possible  that  a  few 
parts  of  iron  per  million  parts  of  butter,  present  in  a  finely  divided, 
colloidal  condition  might  be  able  to  transfer  slowly  some  of  the  in- 
closed oxygen  to  any  one  of  the  oxidizable  substances  present.  It 
is  of  course  well  known  that  in  the  presence  of  a  peroxid  such  as 
hydrogen  peroxid  the  iron  will  rapidly  transfer  oxygen  from  the 
peroxid  to  an  oxidizable  substance.  A  transfer  of  oxygen  such  as 
this  is  often  referred  to  as  peroxidase  action  and  the  substance 
through  which  the  transfer  is  brought  about,  the  colloidal  iron  com- 
pound in  this  case,  is  called  a  peroxidase.  It  is  also  well  known,  at 
least  the  statement  is  often  seen  in  the  literature,  that  peroxidases 
can  transfer  oxygen  only  from  peroxids  and  hence  are  inactive  in  the 
absence  of  peroxids.  Kastle,1  in  discussing  the  mode  of  action  of 
peroxidase,  states:  "According  to  Bach  and  Chodat 2  the  peroxidases 
exert  not  the  slightest  oxidizing  action  in  the  absence  of  the  peroxid." 
It  is  perhaps  to  be  regretted  that  this  statement  was  not  accompanied 
with  another  to  the  effect  that  this  conclusion  follows  from  an  experi- 

1  Kastle.  J.  H.  The  oxidases  and  other  oxygen-catalysts  concerned  in  biological  oxidations.  United 
States  Treasury  Department,  Public  Health  and  Marine-Hospital  Service,  Hygienic  Laboratory,  Bulletin 
59.    Washington,  1910.    Seep.  117. 

*  Bach,  A., and  Chodat,  R.  Ueber  Peroxydase.  Berichte  der  Deutschen Cheraischen  Gesellschaft,  vol. 
36,  no.  3,  pp.  600-605.    Berlin,  Feb.  21, 1903. 


56  CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 

ment  in  which  Bach  and  Chodat '  made  observations  on  oxygen  ab- 
sorption for  a  period  of  24  hours.  Bach  and  Chodat,2  in  support  of 
their  contention  that  peroxidases  are  without  action  in  the  absence 
of  peroxids,  quote  the  work  of  Linossier 3  which  was  likewise  a  series 
of  observations  on  peroxidase  action  covering  only  very  short  periods 
of  time.4  . 

It  may  be  true  that  peroxidases  in  the  presence  of  air  are  inactive 
when  their  activities  are  measured  for  intervals  of  24  hours.  But  in 
the  case  of  cold-storage  butter,  in  which  iron  and  air  may  interact  for 
several  months,  the  possibility  that  oxidative  action  may  take  place 
is  not  excluded,  in  spite  of  the  fact  that  there  is  at  present  no  reason 
to  suppose  that  peroxids  are  present  in  butter. 

The  experiments  here  described  on  the  oxidation  of  lactose  in  milk 
through  the  action  of  iron  salts  are  in  many  respects  similar  to  those 
made  in  recent  years  by  several  investigators  whose  work  throws  some 
light  on  the  subject. 

Lob  and  Pulvermacher 5  studied  the  action  of  gaseous  oxygen  and 
of  hydrogen  peroxid  on  dextrose  and  sucrose.  They  used  as  an 
oxidative  agent  a  preparation  made  by  extracting  pig  pancreas  with 
alcohol  and  adding  iron  to  the  alcohol  nitrate.  The  precipitate  of 
iron-pancreatic  material  was  dried  and  used  in  the  experiments.  In 
24  to  48  hours  this  substance  could  oxidize  dextrose  in  aqueous  solution 
either  in  the  presence  of  hydrogen  peroxid  or  by  the  aid  of  a  stream 
of  air  or  oxygen.  Sucrose  was  also  oxidized  by  hydrogen  peroxid 
and  iron-pancreas  powder,  but  to  a  much  less  extent.  They  sug- 
gest the  necessity  of  the  inversion  of  the  sugar  before  oxidation  can 
take  place. 

Battelli  and  Stern6  and  Harden  and  MacLean  7  studied  oxidation 
in  isolated,  hashed  animal  tissues.  These  investigators  used  such 
material  as  hashed  muscle,  suspended  in  water.  The  suspensions 
were  placed  in  a  flask  filled  with  gaseous  oxygen.  The  substance  to 
be  oxidized  was  added  to  the  suspension  and  the  flasks  were  connected 
with  an  apparatus  for  measuring  the  amount  of  oxygen  absorbed. 

According  to  Battelli  and  Stern,  succinic  acid  is  very  easily  oxidized 
in  a  very  few  hours  under  the  conditions  of  their  experiment.  Harden 
and  MacLean  repeated  some  of  the  experiments  of  Battelli  and  Stern 

1  Bach,  A.,  and  Chodat,  R.    Loc.  cit.    See  pp.  604-605. 

s  Bach,  A.,  and  Chodat,  R.    Loc.  cit.    See  p.  605. 

•Linossier,  G.  Contribution  &  l'£tude  des  ferments  oxydants.  Comptes  Rendus  Hebdomadaires  des 
Seances  de  la  Soctete  de  Biologie,  vol.  50,  no.  12,  pp.  373-375.    Paris,  Apr.  1, 1898. 

*  The  reference  to  Linossier  given  by  Bach  and  Chodat  is  incorrect;  the  reference  by  Kastle  is  correct. 

5  Lob,  Walther,  and  Pulvermacher,  Georg.  Uber  dieoxydative  ZuckerzerstorungunterderEinwirkung 
von  Organpraparaten.    Biochemische  Zeitschrift,  vol.  29,  no.  4/5,  pp.  316-346.    Berlin,  Nov.  22, 1910. 

« Battelli,  F.,  and  Stern,  L.  Die  oxydation  der  Bernsteinsaure  durch  Tiergewebe.  Biochemische 
Zeitschrift,  vol.  30,  no.  1/2,  pp.  172-194.    Berlin,  Dec.  23, 1910. 

7  Harden,  Arthur,  and  MacLean,  Hugh.  The  oxidation  of  isolated  animal  tissues.  Journal  of  Physiol- 
ogy, vol.  43,  no.  1,  pp.  34-45,  Sept.  11, 1911. 


THEORETICAL   CONSIDERATIONS.  57 

and  found  that  the  oxidation  of  succinic  acid  was  not  as  vigorous  as 
Battelli  and  Stern  had  found. 

There  are  several  reasons  why  the  results  on  peroxidase  action 
obtained  by  one  investigator  might  not  be  the  same  as  those  obtained 
by  another  on  material  intended  to  represent  exactly  the  material  pre- 
viously used.  The  activity  of  peroxidases  is  influenced  by  so  many 
conditions  that  an  exact  reproduction  of  any  particular  mixture  is 
perhaps  more  difficult  than  might  at  first  be  supposed.  Furthermore, 
according  to  Wolff,1  the  iron  peroxidase  is  very  specific  in  its  action, 
its  specificity  being  determined  by  the  other  substances  which  may  be 
present.  Certain  iron  salts  or  combinations  of  such  may  oxidize  one 
phenol  and  be  incapable  of  oxidizing  any  other. 

The  results  obtained  in  experiments  on  the  oxidation  of  such  sub- 
stances as  dextrose  must  be  interpreted  carefully,  as  Levene  and 
Meyer 2  have  pointed  out.  •  They  showed  that  in  a  sugar  solution  the 
reducing  power  of  which  had  been  lowered  by  the  combined  action  of 
muscle  plasma  and  pancreatic  extract  the  reducing  power  was  re- 
stored to  its  original  height  by  boiling  under  a  return  condenser  for  two 
hours  in  the  presence  of  1  per  cent  hydrochloric  acid  and  that  a  sub- 
stance having  the  properties  of  a  biosazone  could  be  obtained  from 
the  above  described  solution.  So  that  loss  of  reducing  power  does 
not  necessarily  imply  destruction  of  sugar;  it  may  mean  a  simple 
polymerization. 

The  results  of  the  above-mentioned  investigations  were  used  in 
planning  the  experiments  that  follow: 

THE  OXIDATION  OF  LACTOSE  IN  BUTTER. 

For  the  purpose  of  ascertaining  whether  lactose  which  is  ordinarily 
present  in  butter  to  the  extent  of  about  0.1  to  0.2  per  cent  is  being 
oxidized  in  cold-storage  butter,  with  the  production  of  substances 
having  a  disagreeable  taste  or  smell,  any  one  of  several  methods  sug- 
gest themselves  as  possible.  The  lactose  present  in  a  lot  of  butter 
before  and  after  storage  may  be  estimated  by  any  one  of  the  well- 
known  methods,  or  the  oxidation  products  of  lactose  may  be  looked 
for  in  storage  butter. 

But  the  possible  oxidation  of  lactose  with  the  formation  of  off 
flavors  might  be  brought  about  with  such  little  change  in  the  lactose 
content  that  the  ordinary  methods  of  analysis  might  be  inadequate  for 
the  detection  of  the  change.  It  was  pointed  out  before  (p.  6)  that 
very  small  amounts  of  some  substances  are  easily  detected  by  the 
senses  of  taste  and  smell  and  that  these  amounts  are  smaller  than  are 

1  Wolff,  J.  Relations  entre  les  phenomenes  oxydasiques  naturels  et  artiflciels.  Annales  de  l'Institut 
Pasteur,  vol.  24,  no.  10,  pp.  789-797.    Paris,  Oct.  25,  1910. 

J  Levene,  P.  A.,  and  Meyer,  G.  M.  On  the  combined  action  of  muscle  plasma  and  pancreas  extract  on 
glucose  and  maltose.    Journal  of  Biological  Chemistry,  vol.  9,  no.  2,  pp.  97-107.    Baltimore,  April,  1911. 


58  CHANGE  IN   FLAVOR  OF   STORAGE  BUTTER. 

detectable  by  the  best  analytic  methods  of  the  present  time.  It  was 
also  pointed  out  (p.  15)  that  the  separation  of  fat  quantitatively 
from  butter  is  difficult.  This  introduces  a  difficulty  in  the  direct 
estimation  of  lactose  in  the  butter  curd  solution. 

For  these  and  still  other  reasons  it  was  considered  advisable  to 
avoid  attempting  to  detect  directly  very  minute  changes  in  the 
lactose  content  of  storage  butter.  The  problem  was  approached 
indirectly. 

On  account  of  the  presence  of  sodium  chlorid  in  butter  in  amounts 
varying  from  about  12  to  over  30  per  cent  in  the  curd  solution,  care 
must  be  taken  in  applying  the  results  of  other  investigators  on 
peroxidases  to  this  problem.  It  may  be  that  the  sodium  chlorid  is 
without  effect.  Obviously,  only  experimental  data  in  which  the  per- 
oxidase action  in  the  presence  of  sodium  chlorid  is  studied  are  directly 
applicable  here. 

One  method  -used  in  studying  (sample  No.  25)  the  utilization  of 
oxygen  by  iron  in  butter  was  similar  in  some  respects  to  that  used  by 
Horbaczewski *  and  others  in  their  studies  of  the  utilization  of  atmos- 
pheric oxygen  by  oxidases  of  animal  tissues. 

Description  of  samples. — Several  gallons  of  raw  milk  were  obtained 
from  a  dealer  and  separated  in  the  cream  separator  in  the  laboratory. 
To  8  liters  of  skim  milk  sodium  chlorid  was  added,  in  the  proportion 
of  180  grams  of  sodium  chlorid  to  1  liter  of  skim  milk.  Two  and  one- 
half  liters  of  this  sample  (milk  No.  25)  were  transferred  to  an  8-liter 
flask  provided  with  a  rubber  stopper  through  which  an  inlet  and  outlet 
glass  tube  passed  to  permit  the  passage  of  the  oxygen  gas  through  the 
sample.  Part  of  the  sample  was  set  aside.  The  milk  in  the  flask  was 
the  material  on  which  the  experiment  was  made. 

Methods  and  experimental  procedure. — Before  beginning  the  experi- 
ment the  lactose  content  of  the  sample  was  determined  by  its  reducing 
power  and  with  the  polariscope.  In  so  far  as  no  very  great  change  in 
this  quantity  was  expected,  the  greatest  care  was  taken  to  obtain 
accurate  results  and  uniformity  of  procedure. 

For  the  gravimetric  estimation  of  lactose  the  method  described  on 
pages  42,  48,  and  119,  Bulletin  107  (revised),  of  the  Bureau  of  Chem- 
istry was  used.  It  is  of  course  questionable  whether  the  amount  of 
copper  reduced  by  a  given  weight  of  lactose  in  milk  will  be  the  same 
as  that  reduced  by  the  same  weight  of  lactose  in  milk  containing  18 
per  cent  of  sodium  chlorid.  But  what  was  sought  was  not  the  abso- 
lute amount  of  lactose  present,  but  rather  the  difference,  if  any, 
between  the  amount  of  lactose  present  before  and  after  a  certain 
treatment  of  the  sample. 

1  Horbaczewski,  J.    Untersuchungen  ttber  die  entstehung  der  Harnsaure  im  Saugethier  organismus. 
Monatshefte  fiir  Chemie,  vol.  10,  pp.  624-641.    Vienna,  1889. 


THEORETICAL  CONSIDERATIONS.  59 

Six  Gooch  crucibles  were  prepared  and  used  as  described  by 
Kendall.1 

The  weights  of  the  Gooch  crucibles  after  a  determination  were 
less  than  their  weights  before  by  an  amount  that  varied  between  0.4 
and  1.2  milligrams.  The  average  loss  in  weight  for  19  determinations 
was  0.8  milhgram.  Only  once  did  a  crucible  show  a  gain  in  weight, 
0.3  milligram.  ^ 

Although  reagents  (copper  sulfate,  sodium  potassium  tartrate, 
sodium  hydroxid)  of  the  highest  purity  were  used,  a  slight  reduction 
was  always  obtained  in  blank  experiments  made  on  an  18  per  cent 
aqueous  solution  of  sodium  chlorid.  The  weight  of  cuprous  oxid 
obtained  in  the  blank  determinations  (9)  varied  from  3.6  to  9.1 
milligrams,  average  5.9  milligrams. 

The  reducing  power  of  sample  No.  25  was  determined  six  times  in 
duplicate  during  the  course  of  the  experiment.  The  duplicates  dif- 
fered by  the  following  weights  of  cuprous  oxid:  4.3,  0.4,  2.1,  0.2,  0.4, 
0.8  milligrams.  Very  nearly  378  milligrams  of  cuprous  oxid  were 
always  obtained  in  a  determination,  from  which  amount  the  amount 
of  the  blank  determination  was  subtracted. 

For  the  determination  of  lactose  by  the  polariscope,  the  method 
given  in  Bulletin  107  (revised),  Bureau  of  Chemistry,  pages  118-119, 
was  used:  Eight  cubic  centimeters  of  acid  mercuric  nitrate  was  used 
as  the  precipitant  for  131.6  grams  of  the  sample  in  a  200  cubic  centi- 
meter flask.  The  filtrate  was  polarized  in  a  400-millimeter  tube  in  a 
Ventzke  polariscope.  The  readings  divided  by  4  give  per  cent  of 
lactose,  if  it  be  assumed  that  the  sodium  chlorid  was  without  effect  on 
the  rotatory  power  of  the  lactose.  The  results  are  collected  in  Table  19. 
Lactose  containing  one  molecule  of  water  of  crystallization  calculated 
from  the  polariscope  readings  was  present  to  the  extent  of  4.75  per 
cent.  The  average  weight  of  cuprous  oxid  found,  0.371  gram,  corre- 
sponds to  0.2577  gram  of  lactose  or  5.19  grams  of  lactose  in  100  cubic 
centimeters  sample  No.  25. 

The  nitrate  from  the  mercuric  nitrate  precipitation,  although  per- 
fectly clear  at  first,  soon  becomes  cloudy  and  unfit  for  reading  in 
the  polariscope.  However,  readings  can  be  taken  without  interfer- 
ence by  cloudiness  if  they  are  taken  within  one-half  hour  after  filtering. 
The  polariscope  readings  of  the  filtrates  were  found  to  undergo  no 
change  even  after  being  allowed  to  remain  in  the  laboratory  for  two 
weeks.  After  adding  the  precipitant  and  diluting  to  the  mark  the 
mixture  was  allowed  to  stand  one  hour.  It  was  then  filtered  and  the 
filtrate  immediately  polarized.  Several  days  after,  the  filtrate  was 
again  filtered,  a  precipitate  having  formed  in  the  meantime,  and 
polarized.     In  this  way  several  filtrates  were  repeatedly  polarized  at 

1  Kendall,  Edward  Calvin.    A  quantitative  study  of  the  action  of  pancreatic  amylase.    Columbia  Uni- 
versity, Dissertation,  1910.    See  p.  10. 


60  CHANGE   IN   FLAVOR   OF   STORAGE  BUTTER. 

intervals  of  a  few  days  without  in  any  case  detecting  an  appreciable 
variation  in  polariscope  reading. 

After  having  determined  the  lactose  in  the  sample  No.  25,  its 
specific  gravity  (in  a  25  cubic  centimeter  pycnometer)  was  determined. 
Oxygen  was  then  passed  through  the  sample  for  72  hours.  The  oxygen 
was  passed  from  the  oxygen  tank  into  an  8-liter  flask  containing  3  liters 
of  18  percent  sodium chlorid  solution  and  then  through  the  milk.  This 
was  done  so  that  the  gas  as  it  slowly  bubbled  through  the  milk  would 
alter  the  concentration  of  the  sample  as  little  as  possible.  While 
the  contact  between  gas  and  liquid  was  poor,  it  is  almost  certain  that 
the  liquid  was  saturated  with  the  gas.  About  25  gallons  (not  quite 
100  liters)  of  oxygen  passed  through  the  sample  in  the  72  hours. 
Before  each  determination  of  lactose  the  specific  gravity  of  the  sample 
was  determined  and  recorded.     No  significant  variations  were  observed. 

After  the  72  hours'  passage  of  the  oxygen  gas  the  lactose  content 
of  the  sample  was  determined  and  found  to  have  undergone  no 
change.  This  showed,  at  least  in  this  particular  case,  that  the 
naturally  occurring  peroxidase  in  milk  could  not  utilize  molecular 
oxygen  for  the  oxidation  of  lactose.  From  time  to  time  tests  for 
peroxidase  were  made,  using  tincture  of  guaiac  and  a  few  drops  of  a 
dilute  solution  of  hydrogen  peroxid.  Peroxidase  was  present  in  the 
material  throughout  the  experiment.  Although  always  looked  for, 
oxidase  was  not  found  in  the  several  tests,  except  once,  and  that  was 
probably  due  to  some  unaccountable  error.  The  reagent,  tincture 
of  guaiac,  was  not  the  cause  of  the  unusual  positive  test. 

To  sample  No.  25  there  was  then  added  8  grams  of  ferrous  sulfate 
containing  7  molecules  of  water  of  crystallization  (FeS047H20).  One 
gram  of  metallic  iron  is  present  in  4.978  grams  of  this  salt.  On  adding 
ferrous  sulfate  to  the  milk  a  very  strong  disagreeable  odor  was  pro- 
duced, suggesting  putrefying  protein.  The  odor  was  undoubtedly 
produced  by  the  action  of  the  iron  on  the  milk,  as  the  sample  was 
odorless  before  the  addition.  The  odor  of  the  material  throughout 
the  experiment  was  always  carefully  noticed.  (See  p.  64.)  The 
quantity  of  ferrous  sulfate  added  was  calculated  to  make  one  part  of 
metallic  iron  present  in  1,000  parts  of  milk.  This  is  undoubtedry 
much  more  than  is  ever  present  in  milk  or  butter,  excluding  the  case 
where  butter  is  in  contact  with  iron  rust,  which  has  gone  into  solution 
but  has  not  diffused  very  far  into  the  butter  mass.  But  the  amount 
of  iron  was  purposely  made  very  high  because  the  experimental  time 
was  to  be  much  shorter  than  the  storage  period.  The  milk  was  allowed 
to  stand  one  day  after  the  addition  of  the  iron,  and  the  lactose  deter- 
mined. No  change  was  noticed  in  this  quantity  nor  was  any  change 
found  after  blowing  oxygen  through  the  sample  for  a  second  period  of 
72  hours.  The  acidity  of  the  sample  to  phenolphthalein  did  not 
change  during  this  time,  nor  were  bacteria  present  during  the  experi- 


THEORETICAL   CONSIDERATIONS. 


61 


ment  in  numbers  sufficient  to  affect  the  results.  Oxygen  was  passed 
tlirough  the  sample  for  17  days  longer  with  a  decided  drop  in  reducing 
power  and  polarization  at  the  end  of  that  period.  This  was  due  to 
bacterial  action,  as  was  shown  by  a  suitable  examination. 

Table  19. — Action  oj  iron  and  oxygen  on  lactose  in  milk  No.  25. 


No. 


Treatment. 


Reduction. 


Polarization. 


Date  of  deter- 
mination. 


Weight  of 
Cu20. 


Readings 

on  Ventzke 

scale. 


Date  of 


25 
25.1 

25. 2& 
25.2a 
25.2b 

25.3 


Original  sample,  containing  sodium  chlorid; 

age,  7  days 

Repetition  on  same  sample 

After  blowing  oxygen  through  for  72  hours  and 

letting  stand  1  day 

Added  ferrous  sulfate  and  let  stand  1  day 

Repetition  on  part  of  the  sample  set  aside  until . 
After  blowing  oxygen  through  sample  for 

second  period  of  72  hours,  a  total  of  144 

After  10  days  slow  and  7  days  rapid  blowing 

of  oxygen  through  sample 


Mar.  28,1911 
Mar.  30,1911 

Apr.     4,1911 
.do. 


Apr.     7,1911 

....do 

JApr.  25,1911 


Grams. 
0. 3721 
.3720 

.3703 
.3721 
.3716 

.3692 

.2084 


+19.0 
19.0 


18.9 
18.8 


Mar.  28 
Mar.  30 

Apr.     3 
Apr.     5 


18.6 
17.7 
13.2 


Apr.  8 
Apr.  17 
Apr.  25 


During  the  summer  of  1910  three  experiments,  samples  Nos.  1,  2, 
and  3,  essentially  similar  to  that  on  milk  No.  25,  were  made.  Air 
instead  of  oxygen  was  used.  The  results  are  not  appreciably  differ- 
ent from  those  in  Table  19.  Sample  No.  1  was  milk,  samples  2  and  3 
were  skim  milk;  all  three  were  soured  before  use  in  the  experiments. 
Only  the  reduction  was  determined  in  these  samples  which,  because 
of  the  formation  of  lactic  acid,  was  lower  than  the  reduction  in  sample 
No.  25.  But  the  reduction  did  not  change  appreciably  by  the  action 
of  the  air  blown  through  the  samples.  The  results  were  not  as  uni- 
form as  in  Table  19,  very  likely  because  it  is  more  difficult  to  sample 
accurately  the  sour  milk  containing  particles  of  casein.  After  a  few 
trials  during  the  summer  of  1910  on  the  best  way  of  obtaining  a 
uniform  sample  for  reduction,  it  was  found  best  to  transfer  the  portion 
of  the  sample  in  the  pycnometer  (25  cubic  centimeters)  to  the  volu- 
metric flask  for  clarification.  In  this  way  both  the  volume  and  weight 
of  the  portion  used  was  known. 

THE     POSSIBLE    OXIDATION    OF    LACTOSE    IN     STORAGE    BUTTER    BY    A 

PEROXID. 

It  was  stated  before  (p.  56)  that  there  are  at  present  no  reasons  for 
supposing  that  peroxids  are  present  to  any  appreciable  extent  in 
butter.  This  statement  is  not  free  from  assumption,  for  there  is  a 
possibility  of  the  slow  formation  of  an  organic  peroxid  in  butter. 
A  review  of  the  literature  on  the  subject  and  an  interesting  discussion 
of  the  formation  of  organic  peroxids  by  direct  combination  of  the 
compound  with  molecular  oxygen  has  been  made  by  Kastle.1     The 


i  Loc.  cit. 


62  '    CHANGE   IN    FLAVOR   OF   STORAGE  BUTTER. 

detection  of  an  organic  peroxid  in  butter  at  any  particular  time  might 
be  practically  impossible  because  of  its  almost  immediate  decomposi- 
tion either  by  the  catalase  naturally  present  in  unpasteurized  cream 
butter  or  by  iron  almost  always  present,  even  in  butter  most  carefully 
churned,  to  the  extent  of  three  or  four  parts  per  million  of  butter 
(see  p.  54  for  amounts  of  iron  in  butter)  and  yet  the  oxidative  process 
may  be  taking  place.  Perhaps  a  careful  analysis  of  the  gas  in  butter 
will  show  whether  any  of  the  oxygen  is  being  removed  in  this  way. 
The  work  on  the  analysis  of  the  gases  in  butter  has  been  begun  and 
some  of  the  analyses  are  given  on  page  37. 

For  the  reasons  just  mentioned  the  fact  that  no  oxidation  of  lactose 
was  detected  in  skim  milk  containing  iron  and  through  which  oxygen 
was  passed  does  not  exclude  the  possibility  of  the  slow  formation  of 
organic  peroxids  in  butter  and  their  subsequent  oxidative  action. 

On  the  assumption  that  organic  peroxids  might  be  slowly  formed 
in  butter,  and  that  such  peroxids  might  be  used  by  the  peroxidase 
present  for  oxidation,  a  few  experiments  were  made  in  which  the 
polarization  of  skim  milk  was  observed  before  and  after  the  addition 
of  hydrogen  peroxid,  with  and  without  iron.  The  data  are  sum- 
marized in  Table  20.  One  liter  of  each  mixture  was  prepared  from 
which  portions  were  transferred  to  a  200  cubic  centimeter  flask  for 
clarification  and  polarization. 

It  is  evident  that  in  these  mixtures  containing  hydrogen  peroxid 
and  iron  (Nos.  18,  19,  and  23)  there  was  a  very  appreciable  lowering 
in  the  polarization.  This  lowering  probably  was  not  due  to  the  easy 
reducibility  of  the  mercury  by  the  lactose,  because  in  those  filtrates 
containing  hydrogen  peroxid  but  no  iron  (Nos.  17  and  21)  there  was 
no  lowering,  although  these  filtrates  contained  unprecipitated  mer- 
cury as  well  as  the  others.  While  there  may  be  other  possibilities, 
the  most  reasonable  tentative  conclusion  to  be  drawn  from  the  results 
of  Table  20  is  that  the  lactose  was  oxidized  by  the  action  of  the 
peroxid  and  iron,  and  if  there  were  peroxid  formation  in  butter  such 
oxidation  of  lactose  might  take  place  there.  The  experiments  detailed 
in  Table  20  were  not  expected  to  be  conclusive;  others  are  undoubtedly 
necessary. 


THEORETICAL   CONSIDERATIONS. 


63 


s    . 


6-d 


OJ  OCO.S5 


oioui 


C»COt~-  CO 


eoeocoi^ 


.2  o  o  o  o 


o  .2  •£  .2 

3- OS 


all 

■a  &— 

CO  3 


H 


Sec  as 
g  o  o  o 
SZZZ 

5 


o  o 

OZZ 


.88SS 


tiz 


.0.0 


.fi.fi 


:  a 

113  03 

'.•CO 

,-  S  e 

£  -^  <i>  a  — ' 

Pl,    .Ha; 


B-d 
.2"C 


£  x  0.5 
>>fe  So 


llj 


Bl 


NOrea. 
oi  oi  oi  c-i 


oi  o>  oi  ec 


NOMO 
OS  OS  ai  -1" 


OSOC5CC 
30  OS  OS  C» 


os  os  oi  ci 


Ofnoa 


.£  o  o  o  o 
|ZZIZZ 


~  -  - 

SCC- 
ISO!) 


fc     SB 


!§§§ 


1   6 


«    § 


64  CHANGE  IN   FLAVOR   OF   STORAGE   BUTTER. 

Lob  and  Pulvermacher's  observation  (p.  56)  that  dextrose  is  much 
more  easily  oxidized  than  sucrose,  and  that  the  sucrose  apparently 
can  oxidize  only  as  fast  as  it  is  first  inverted,  suggests  the  desirability 
of  further  experiment  along  the  lines  of  the  work  on  sample  No.  25, 
but  in  which  the  sample  is  soured  before  being  used  in  the  experiment. 
In  the  presence  of  lactic  acid  (or  its  combination  with  casein)  it  is 
very  probable  that  the  lactose  present  would  be  inverted,  even  if  very 
slowly.  Then  the  iron  and  oxygen  would  have  the  entire  storage 
period  of  several  months  to  bring  about  the  slight  chemical  changes 
presumably  sufficient  to  give  butter  an  "off  flavor." 

ODORS    PRODUCED    IN    MILK    BY    THE    ADDITION    OF    IRON    SALTS. 

In  our  experiments  the  production  of  substances  having  a  dis-* 
agreeable  odor  and  taste  was  the  most  important  part  of  the  work. 
No  chemical  change,  however  pronounced,  that  presumably  did  not 
affect  the  flavor  of  butter  was  of  more  than  incidental  interest. 
For  this  reason  the  odor  of  the  experimental  material  was  always 
carefully  noted. 

It  was  stated  before  (p.  60)  that  on  adding  ferrous  sulfate  to  milk 
containing  salt,  a  very  strong,  nauseating  odor  was  produced.  This 
observation  had  been  made  before  in  mixtures  of  milk,  hydrogen 
peroxid  and  ferric  chlorid,  but  the  odor  was  not  produced  every  time 
that  ferric  chlorid  and  hydrogen  peroxid  were  added  to  milk.  It 
was  obviously  desirable  to  know  whether  iron  salts  could  produce 
undesirable  odors  in  milk  and  whether  the  experimental  conditions 
under  which  such  odors  were  produced  were  in  any  way  similar  to  the 
conditions  in  cold  storage  butter. 

After  several  trials  it  was  found  that  ferrous  salts  added  to  milk 
in  the  proportion  of  1  part  of  metallic  iron  to  1,000  parts  of  milk 
would  result  in  the  production  of  a  very  powerful  odor.  The  method 
of  the  experiment  was  very  simple  as- the  following  example  shows: 

Several  liters  of  raw  milk  as  obtained  fresh  from  the  dealer  were 
separated.  Sodium  chlorid  was  added  to  the  skim  milk,  anywhere 
from  18  per  cent  to  saturation,  or  the  sodium  chlorid  may  be  omitted 
altogether.  The  sodium  chlorid  serves  both  as  a  preservative  and  as  a 
normal  constituent  of  butter  curd  solution.  Skim  milk  was  used 
because  by  eUminating  the  fat  and  fat  soluble  substances  the  sub- 
stances causing  the  odor  could  be  better  determined.  The  sample 
of  salted  skim  milk  could  be  used  at  once  or  after  several  days'  stand- 
ing at  room  temperature.  Portions  of  300  cubic  centimeters  each 
were  transferred  to  1-liter  Erlenmeyer  flasks.  To  each  flask  there 
was  then  added  a  calculated  quantity  of  the  metal  salt.  The  follow- 
ing salts  were  used  in  the  several  experiments;  some  of  them  were 
used  very  many  times:   Ferrous  sulfate,  ferrous  ammonium  sulfate, 


THEORETICAL   CONSIDERATIONS.  65 

ferrous  lactate,  ferric  acetate,  ferric  chlorid,  and  to  a  lesser  extent 
some  salts  of  the  following  metals:  Copper,  manganese,  aluminium, 
and  lead. 

In  no  case  was  any  odor  produced  by  any  metal  salt  other  than 
iron.  Ferrous  salts  almost  always,  ferric  salts  very  seldom,  pro- 
duced an  odor  in  the  milk.  The  quantities  used  were  calculated  to 
make  1  part  of  metal  present  in  1,000  parts  of  milk.  When  this 
quantity  of  a  ferrous  salt  is  used  the  odor  becomes  very  powerful 
in  a  few  minutes.  However,  1  part  of  ferrous  iron  in  50,000  parts  of 
milk  could  easily  be  shown  to  produce  an  odor  plainly  perceptible  by 
several  persons  to  whom  no  information  had  been  previously  given 
regarding  the  nature  of  the  samples  to  be  smelled.  The  odor  develops 
slowly  when  the  amount  of  iron  is  small.  For  1  part  of  iron  to  50,000 
of  milk  an  hour  should  be  allowed. 

It  seems  that  the  odor  is  developed  at  that  time  when  the  color  of 
the  mixture  changes  to  the  more  highly  colored  ferric  salt. 

Although  the  odor  strongly  suggests  putrefying  protein  or  hydrogen 
sulfid,  tests  made  for  hydrogen  sulfid  were  negative.  The  tests  were 
made  by  passing  a  current  of  air  through  milk  in  which  an  odor  had 
been  developed  by  the  addition  of  ferrous  iron,  and  then  through  an 
alkaline  solution  of  lead  acetate. 

It  is  doubtful  whether  the  odor  came  from  the  fat,  because  skim  milk 
was  used,  and  it  was  doubtful  whether  lactose  was  the  cause.  It 
seemed  probable  that  protein  was  being  acted  upon  by  the  iron.  In 
one  experiment  in  which  solutions  of  egg-white  and  of  egg-yolk  were 
used  the  same  results  were  obtained  as  before,  i.  e.,  the  addition  of 
iron  salts  (ferrous  sulfate  and  ferric  chlorid)  resulted  in  the- pro- 
duction of  a  strong  odor.  Whether  protein  was  acted  upon  in  the 
experiments  or  not,  is  difficult  to  say. 

On  looking  through  the  literature  on  the  oxidation  of  proteins, 
very  little  was  found  that  would  throw  light  on  the  oxidation  of 
protein  by  means  of  comparatively  mild  oxidizing  agents.  In  most 
of  the  researches  the  protein  was  broken  down  completely  by  the 
reagents  used,  and  the  work  was  done  for  the  purpose  of  studying  either 
the  products  of  the  oxidation  or  the  various  oxidation  stages  through 
which  the  protein  goes  during  the  course  of  metabolism.  No  records 
were  found  in  which  the  protein  oxidation  was  studied  for  the  par- 
ticular purpose  of  observing  whether  odoriferous  substances  were 
formed.  The  oxidation  of  protein  in  storage  butter  (if  it  occurs  at 
all)  is  probably  very  mild.  There  seems  to  be  no  apparent  change  in 
the  quantity  of  protein  in  butter  before  and  after  storage. 

However,  two  researches  were  found  in  the  literature  which  were 
highly  instructive. 


66  CHANGE   IN    FLAVOR   OF   STORAGE   BUTTER. 

Neuberg  and  Blumenthal l  studied  the  oxidation  products  of 
gelatin,  using  ferrous  sulfate  and  hydrogen  peroxid.  In  the  distillates 
from  2  kilograms  of  gelatin  they  isolated  and  identified  isovaleralde- 
hyde.  Other  volatile  products  were  also  formed.  Orgler 2  repeated 
some  of  the  work  of  Neuberg  and  Blumenthal,  using  crystallized 
egg  albumin,  copper  sulfate,  and  hydrogen  peroxid.  Acetone  was 
detected  and  identified  in  the  distillates  from  such  a  mixture,  which 
distillate,  according  to  Orgler,  had  a  strong  fruity  odor. 

It  follows  that  if  the  ferrous  salts  used  in  the  production  of  an  odor 
in  milk  act  on  some  milk  protein  with  the  formation  of  aldehyde  or 
ketone  substances,  it  ought  to  be  possible  to  take  milk  (containing  salt) 
add  a  ferrous  salt,  and  obtain  from  it  by  distillation  some  of  the  sub- 
stances mentioned  by  Neuberg  and  Blumenthal  and  by  Orgler.  Two 
experiments  were  made  which,  while  not  so  conclusive  as  to  make 
further  experiment  unnecessary,  gave  results  so  much  in  accord  with 
expectations  that  there  is  little  doubt  but  what  the  ferrous  sulfate 
used  in  the  experiments  caused  the  formation  of  substances  which 
gave  the  iodoform  test.  Beyond  this  their  chemical  nature  was  not 
investigated. 

THE    PRODUCTION    OF    IODOFORM-REACTING    SUBSTANCES    IN    MILK    BY 

FERROUS    IRON. 

To  an  ordinary  2-liter  side-arm  distillation  flask  1  liter  of  fresh 
skim  milk  was  transferred  (sample  42.2).  The  sample  contained  200 
grams  of  sodium  chlorid  to  1  liter  of  skim  milk.  It  was  quickly 
brought  to  a  boil  and  10  cubic  centimeters  distilled  over.  This  was 
tested  with  sodium  hydroxid  solution  (1:3)  and  iodin  solution 
without  heating  for  substances  giving  the  iodoform  test.3 

The  test  was  positive.  The  above  procedure  was  repeated  on 
some  of  the  same  sample  of  milk  under  the  same  conditions,  except 
that  5  grams  of  the  crystallized  ferrous  sulfate  (Fe  1 :  1  000)  was 
added  just  before  distilling.  The  distillate  gave  a  much  stronger 
iodoform  test;  that  is,  there  was  a  very  appreciable  immediate  pre- 
cipitate of  iodoform.  A  second  sample  of  skim  milk  was  distilled 
as  before.  This  sample  (No.  37x)  was  obtained  from  the  same 
dealer  as  the  previous  sample  (sample  42),  and  after  being  skimmed 
they  probably  did  not  differ  very  materially  in  their  composition. 
At  the  time  of  the  experiment  this  sample  (37x)  was  raw  skim  milk 
containing  30  per  cent  of  sodium  chlorid  (300  grams  of  salt  to  1  liter 
of  milk)  and  1  part  of  iron  as  ferrous  sulfate  to  5  000  parts  ol  milk. 

1  Neuberg,  C,  and  Blumenthal,  F.  Uber  die  Bildung  von  Isovaleraldehyd  und  Aceton  aus  Gelatine. 
Beitrage  zur  Chemischen  Physiologie  und  Pathologie,  vol.  2,  no.  5-6,  pp.  238-250.  Braunschweig,  May, 
1902. 

1  Orgler,  Arnold.  Uber  die  Entstehung  von  Aceton  aus  krystallisiertem  Oralbumin.  Beitrage  zur 
Chemischen  Physiologie  und  Pathologie,  vol.  1,  no.  10-12,  pp.  583.    Braunschweig,  January,  1902. 

'  MuUiken,  Samuel  Parsons.    Identification  of  Pure  Organic  Compounds.    See  vol.  1,  p.  166. 


THEORETICAL  CONSIDERATIONS.  67 

The  salt  and  iron  were  added  on  the  same  day  the  sample  was  received. 
After  remaining  at  room  temperature  in  the  laboratory  for  12  days, 
during  which  time  several  liters  were  used  for  other  purposes,  3 
liters  of  this  sample  were  transferred  to  a  7-liter  bottle  and  oxygen 
was  blown  through  it  slowly  for  4  days  (96  hours).  One  liter  of 
this  was  distilled.  The  distillate  (10  c.  c.)  yielded  more  iodoform 
than  either  of  the  other  two.  Judging  by  inspection,  on  distilling 
fresh  skim  milk  a  very  small  but  distinctly  perceptible  quantity  of 
lodoform-reacting  substance  was  obtained;  fresh  skim  milk  and 
iron  yielded  more,  and  skim  milk  and  iron  first  saturated  with  oxygen 
yielded  most.  There  could  be  little  doubt  about  the  relative  amounts 
of  iodoform,  but  the  experiment  was  repeated  on  portions  of  the 
same  samples  as  before  for  the  purpose  of  estimating  quantitatively 
the  iodoform  obtained. 

Each  distillation  was  continued  until  six  10  cubic  centimeter 
portions  of  distillate  were  obtained.  Iodoform  tests  on  these  were 
made  by  adding  to  each  portion  contained  in  a  test  tube  10  drops 
of  sodium  hydroxid  solution  (1:3)  and  sufficient  iodin  solution  to 
insure  a  slight  excess.  In  every  case  precipitates  of  iodoform  were 
obtained  almost  immediately  and  without  the  aid  of  heat.  Very 
little  iodoform,  if  any,  was  obtained  in  the  last  10  cubic  centimeters 
of  distillate. 

The  iodoform  was  washed  first  by  decantation,  then  transferred 
to  filter  papers  and  washed  till  the  filtrates  gave  only  a  very  faint 
cloud  with  silver  nitrate  solution.  Some  of  the  iodoform  was,  of 
course,  lost  through  solution  in  the  wash  water,  but  the  relative 
amounts  in  this  case  rather  than  the  absolute  were  just  as  desirable. 
The  amount  lost  in  this  way  probably  was  small  compared  with  the 
amounts  present.  The  blank  determination  (No.  42.3)  was  lost. 
It  contained  a  distinctly  perceptible  precipitate,  but  too  small  in 
amount  to  compare  with  either  of  the  other  two.  For  the  estima- 
tion of  the  iodoform  a  method  given  in  the  Pharmaceutical  Journal, 
page  555,  volume  82,  1909,  was  used.  The  iodoform  on  the  filter 
papers  was  dissolved  in  alcohol  and  ether  and  the  solutions  received 
in  300  cubic  centimeter  Erlenmeyer  flasks.  To  these  flasks  and  to 
controls  1  cubic  centimeter  nitrous  acid  (fuming  nitric  acid)  and 
50  cubic  centimeters  approximately  tenth  normal  silver  nitrate 
solution  were  added.  The  mixtures  were  heated  on  the  steam  bath 
over  night.  The  silver  still  remaining  in  solution  was  estimated  with 
standard  ammonium  sulfocyanid  solution,  using  ferric  ammonium 
sulfate  as  indicator. 

The  distillate  from  the  fresh  milk  containing  ferrous  sulfate  (No. 
42.4,  Fe  1  :  1,000)  yielded  15.4  milligrams  of  iodoform;  the  distillate 
from  the  12  days'  old  milk  containing  ferrous  sulfate  (No.  37x,  Fe  1 : 
5  000)  and  saturated  with  oxygen  gave  54.7  milligrams  of  iodoform. 


68  CHANGE  IN   FLAVOR   OF   STORAGE  BUTTER. 

These  amounts  of  iodoform  correspond  in  the  titrations  to  1.37 
cubic  centimeters  in  the  first  and  to  4.88  cubic  centimeters  of  silver 
nitrate  solution  (N/11.7)  in  the  second  determination. 

While  these  results  do  not  prove  that  the  iodoform  was  obtained 
from  oxidation  products  of  milk  protein,  they  do  prove  the  possibility 
of  such  oxidation.  By  distilling  such  mixtures  under  low  pressure 
and  at  low  temperature  to  remove  the  possible  objection  that  the 
temperature  of  distillation  is  not  the  temperature  at  which  chemical 
changes  take  place  in  storage  butter,  the  identity  of  the  iodoform- 
reacting  substances  could  without  doubt  be  ascertained. 

Whether  the  small  amounts  of  iron  ordinarily  present  in  butter 
(see  p.  54)  can  slowly  bring  about  the  same  kind  of  a  change  that 
larger  amounts  of  iron  bring  about  in  milk  in  a  very  much  shorter 
time  is  to  be  determined  by  future  investigation. 

SUMMARY. 

The  failure  of  previous  investigators  to  find  evidences  of  pro- 
teolysis in  cold-storage  butter  may  have  been  due  to  difficulty  in 
obtaining  proper  precipitations  in  the  curd  solution. 

Methods  of  analysis  have  been  perfected  which  permit  the  use  of 
large  samples  and  show  the  first  stages  in  the  proteolysis.  This 
method  gave  no  evidence  of  an  increase  in  soluble  nitrogen  in  butter 
on  long  standing  at  0°  F.,  even  when  the  conditions  of  the  manu- 
facture were  most  favorable  to  such  changes. 

Buttermilk  from  sweet  unpasteurized  cream  and  from  sweet 
pasteurized  cream  when  preserved  with  18  per  cent  sodium  chlorid 
to  correspond  to  butter-curd  solution  showed  no  proteolysis  during 
a  long  period  in  cold  storage. 

Bacterial  enzym  held  in  cold  storage  in  milk  containing  18  per 
cent  of  sodium  chlorid  gave  some  evidence  of  proteolysis.  The  action 
of  pepsin  and  trypsin  under  similar  conditions  was  not  completely 
inhibited. 

Butter  made  from  sweet  pasteurized  cream  keeps  much  better 
than  butter  made  from  similar  cream  without  pasteurization,  but 
the  changes  in  the  unpasteurized  cream  butter  can  not  be  repro- 
duced by  reinoculating  the  pasteurized  cream  with  the  bacteria  of 
the  cream  before  pasteurization. 

By  means  of  specially  designed  apparatus  exact  analysis  was 
made  of  the  gases  contained  in  butter.  About  10  per  cent,  by 
volume,  of  fresh  butter  is  gas  consisting  approximately  of  nitrogen 
(by  difference)  33  per  cent,  oxygen  20  per  cent,  and  the  remainder 
of  gases  absorbable  by  sodium  hydroxid.  The  oxygen  was  materially 
less  after  storage. 

The  addition  of  iron  to  the  cream  even  in  as  small  an  amount  as 
one  or  two  parts  per  million  parts  of  cream  has  an  influence  on  the 


SUMMARY. 


69 


flavor  of  the  butter.  This  work  gives  nothing  to  show  that  the 
nature  of  the  flavor  is  appreciably  changed,  but  the  rate  of  develop- 
ment is  accelerated. 

The  cream  may  take  up  iron  in  quantities  sufficient  to  affect  the 
flavor  from  rusty  cans  or  even  from  the  exposed  boltheads  or  other 
metal  parts  of  the  churn. 

The  action  of  copper  is  similar  but  perhaps  more  intense. 

It  was  found  that  in  milk  to  which  18  per  cent  sodium  chlorid 
had  been  added  there  was  no  change  in  the  lactose  when  iron  was 
added  and  a  current  of  oxygen  passed  through  the  milk  for  72  hours. 

A  strong  odor  may  be  produced  in  milk  by  the  addition  of  small 
amounts  of  iron  salts.  The  ferrous  salts  are  more  active  than  the 
ferric  salts. 

The  iodoform  test  is  much  stronger  in  distillates  from  milk  con- 
taining ferrous  sulfate. 


ADDITIONAL  COPIES  of  this  publication 
A  may  be  procured  from  the  Superintend- 
ent of  Documents,  Government  Printing 
OflBce,  Washington,  D.  C,  at  10  cents  per  copy 


.  LIBRARY  FACILITY 


A    001  137  161     4 


